Nucleic acid adaptors and uses thereof

ABSTRACT

Provided herein are compositions, systems, methods, and kits for joining together the ends of one or more polynucleotides using at least one pair of blocking oligonucleotide adaptors. Blocking oligonucleotide adaptors can comprise a double-stranded oligonucleotide adaptor (duplex) having an overhang cohesive portion that anneals with a blocking oligonucleotide which can be a separate single-stranded oligonucleotide. Also provided herein are compositions generated using the blocking oligonucleotide adapters, including circularized polynucleotides, that may be manipulated, for example, through nick translation, to yield a linear mate pair construct.

This application is a Divisional application of U.S. application Ser.No. 13/877,203, having a 35 U.S.C. § 371 filing date of Jun. 3, 2013,now issued U.S. Pat. No. 9,540,637, which is a national stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2011/054053, filed Sep. 29, 2011, which claims the filing datebenefit of U.S. Provisional Application No. 61/389,121, filed on Oct. 1,2010, and 61/426,229, filed on Dec. 22, 2010, and 61/438,259, filed onFeb. 1, 2011, and 61/469,587, filed on Mar. 30, 2011, and 61/498,405,filed on Jun. 17, 2011.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD

The present disclosure relates to compositions, systems, methods andkits for joining the ends of one or more polynucleotides together withone or more oligonucleotide adaptor(s).

INTRODUCTION

Nucleic acid manipulations can often involve joining together the endsof two or more different polynucleotides, or joining together two endsof one polynucleotide for circularization. For example, nucleic acidsample preparation or library preparation workflows can include at leastone joining step mediated by one or more nucleic acid adaptors. Theresulting constructs can be sequenced in a next-generation sequencingprocess, in which large numbers of relatively small nucleic acidfragments can be sequenced at the same time in parallel.

SUMMARY

Provided herein are compositions, systems, methods and kits for joiningtogether the ends of one or more polynucleotides with at least one pairof blocking oligonucleotide adaptors. Blocking oligonucleotide adaptorscan be used to reduce the formation of adaptor dimers or trimers (orhigher-order concatemers) which can improve the yield of desirablepolynucleotide-adaptor products in any recombinant nucleic acidworkflow. A joining step can include joining together two or moredifferent polynucleotides together, or can include joining together twoends of one polynucleotide to form a circular molecule.

Provided herein are nucleic acid compositions, comprising: a firstnucleic acid overhang having a first nucleic acid sequence and a secondnucleic acid overhang having a second nucleic acid sequence, the firstand second nucleic acid sequences can be at least partiallycomplementary to each other, wherein the first overhang can behybridized to a first blocking oligonucleotide over at least a portionof its length and the second overhang can be hybridized to a secondblocking oligonucleotide over at least a portion of its length. In someembodiments, a first nucleic acid overhang can comprise a firstdouble-stranded nucleic acid adaptor. In some embodiments, a secondnucleic acid overhang can comprise a second double-stranded nucleic acidadaptor. In some embodiments, a first or a second double-strandednucleic acid adaptor can comprise biotin.

Provided herein are nucleic acid compositions, comprising: a firstnucleic acid duplex with a first overhang wherein the first overhangincludes a first nucleic acid sequence and a second nucleic acid duplexwith a second overhang wherein the second overhang includes a secondnucleic acid sequence, the first and second nucleic acid sequences canbe at least partially complementary to each other, wherein the firstoverhang can be hybridized to a first blocking oligonucleotide over atleast a portion of its length and the second overhang can be hybridizedto a second blocking oligonucleotide over at least a portion of itslength. In some embodiments, a first nucleic acid duplex can comprise afirst double-stranded nucleic acid adaptor. In some embodiments, asecond nucleic acid duplex can comprise a second double-stranded nucleicacid adaptor. In some embodiments, a first or a second double-strandednucleic acid adaptor can comprise biotin.

Provided herein are nucleic acid compositions, comprising: a firstnucleic acid duplex with a first overhang wherein the first overhangincludes a first nucleic acid sequence and a second nucleic acid duplexwith a second overhang wherein the second overhang includes a secondnucleic acid sequence, the first and second nucleic acid sequences canbe at least partially complementary to each other, wherein the firstoverhang can be hybridized to a first blocking oligonucleotide over atleast a portion of its length and the second overhang can be hybridizedto a second blocking oligonucleotide over at least a portion of itslength, wherein the first nucleic acid duplex can be joined to one endof a first polynucleotide and the second nucleic acid duplex can bejoined to the one end of a second polynucleotide. In some embodiments, afirst and a second polynucleotide can comprise double-strandedpolynucleotides. In some embodiments, a first and a secondpolynucleotide can comprise polynucleotides of interest. In someembodiments, a first or a second double-stranded nucleic acid adaptorcan comprise biotin. In some embodiments, a first and a secondpolynucleotide can be double-stranded polynucleotides and can form partof a single contiguous double-stranded nucleic acid molecule. In someembodiments, a first and a second polynucleotide can be double strandedpolynucleotides and can form part of different double-stranded nucleicacid molecules. In some embodiments, a first nucleic acid duplex cancomprise a first double-stranded nucleic acid adaptor. In someembodiments, a second nucleic acid duplex can comprise a seconddouble-stranded nucleic acid adaptor.

In some embodiments nucleic acid compositions can include a firstblocking oligonucleotide which can comprise a single-strandedoligonucleotide.

In some embodiments nucleic acid compositions can include a secondblocking oligonucleotide which can comprise a single-strandedoligonucleotide.

In some embodiments nucleic acid compositions can include a firstblocking oligonucleotide and a second blocking oligonucleotide that arenot hybridized to each other.

In some embodiments nucleic acid compositions can include a firstoverhang which can be located at an end of a first nucleic acid duplex,a second overhang which can be located at an end of a second nucleicacid duplex, where hybridization of the first overhang to the firstblocking oligonucleotide can form a third nucleic acid duplex, wherehybridization of the second overhang to the second blockingoligonucleotide can form a fourth nucleic acid duplex, and where themelting point of the first nucleic acid duplex and of the second nucleicacid duplex can both be substantially greater than the melting points ofboth the third and fourth nucleic acid duplexes.

In some embodiments nucleic acid compositions can include a firstoverhang which can be located at an end of a first nucleic acid duplex,a second overhang which can be located at an end of a second nucleicacid duplex, where hybridization of the first overhang to the firstblocking oligonucleotide can form a third nucleic acid duplex, wherehybridization of the second overhang to the second blockingoligonucleotide can form a fourth nucleic acid duplex, and wherein themelting point of the first nucleic acid duplex can be substantiallygreater than the melting point of the third nucleic acid duplex.

In some embodiments nucleic acid compositions can include a firstoverhang which can be located at an end of a first nucleic acid duplex,a second overhang which can be located at an end of a second nucleicacid duplex, where hybridization of the first overhang to the firstblocking oligonucleotide can form a third nucleic acid duplex, wherehybridization of the second overhang to the second blockingoligonucleotide can form a fourth nucleic acid duplex, and where themelting point of the second nucleic acid duplex can be substantiallygreater than the melting point of the fourth nucleic acid duplex.

In some embodiments nucleic acid compositions can include a firstnucleic acid overhang which can comprise one end of a firstpolynucleotide and can include a second nucleic acid overhang which cancomprise one end of a second polynucleotide.

In some embodiments, a first and a second polynucleotide can bedouble-stranded polynucleotides and can form part of a single contiguousdouble-stranded nucleic acid molecule.

In some embodiments, a first and a second polynucleotides can be doublestranded polynucleotides and can form part of different double-strandednucleic acid molecules.

Provided herein are methods for joining a first and a second nucleicacid, comprising the step(s): providing a first nucleic acid overhanghaving a first nucleic acid sequence and a second nucleic acid overhanghaving a second nucleic acid sequence, the first and second nucleic acidsequences can be at least partially complementary to each other, whereinthe first overhang can be hybridized to a first blocking oligonucleotideover at least a portion of its length and the second overhang can behybridized to a second blocking oligonucleotide over at least a portionof its length. In some embodiments, the methods can further comprise thestep(s): separating the first blocking oligonucleotides from the firstnucleic acid overhangs. In some embodiments, the methods can furthercomprise the step(s): separating the second blocking oligonucleotidesfrom the second nucleic acid overhangs. In some embodiments, the firstblocking oligonucleotides can be separated from the first nucleic acidoverhangs and the second blocking oligonucleotides can be separated fromthe second nucleic acid overhangs essentially simultaneously orsequentially. In some embodiments, the methods can further comprise thestep(s): hybridizing the first nucleic acid sequence with the secondnucleic acid sequence, thereby joining together the first and the secondnucleic acid overhangs. In some embodiments, a first nucleic acidoverhang can comprise a first double-stranded nucleic acid adaptor. Insome embodiments, a second nucleic acid overhang can comprise a seconddouble-stranded nucleic acid adaptor. In some embodiments, a first or asecond double-stranded nucleic acid adaptor can comprise biotin.

Provided herein are methods for joining a first and a second nucleicacid, comprising the step(s): providing a first nucleic acid duplex witha first overhang wherein the first overhang can have a first nucleicacid sequence and a second nucleic acid duplex with a second overhangwherein the second overhang can have a second nucleic acid sequence, thefirst and second nucleic acid sequences can be at least partiallycomplementary to each other, wherein the first overhang can behybridized to a first blocking oligonucleotide over at least a portionof its length and the second overhang can be hybridized to a secondblocking oligonucleotide over at least a portion of its length. In someembodiments, the methods can further comprise the step(s): separatingthe first blocking oligonucleotides from the first nucleic acidoverhangs. In some embodiments, the methods can further comprise thestep(s): separating the second blocking oligonucleotides from the secondnucleic acid overhangs. In some embodiments, the first blockingoligonucleotides can be separated from the first nucleic acid overhangsand the second blocking oligonucleotides can be separated from thesecond nucleic acid overhangs essentially simultaneously orsequentially. In some embodiments, the methods can further comprise thestep(s): hybridizing the first nucleic acid sequence with the secondnucleic acid sequence to join together the first and the secondoverhangs thereby joining the first and second nucleic acid duplexes. Insome embodiments, a first nucleic acid duplex can comprise a firstdouble-stranded nucleic acid adaptor. In some embodiments, a secondnucleic acid duplex can comprise a second double-stranded nucleic acidadaptor. In some embodiments, a first or a second double-strandednucleic acid adaptor can comprise biotin.

Provided herein are methods for joining a first and a second nucleicacid, comprising the step(s): providing a first nucleic acid duplex witha first overhang wherein the first overhang includes a first nucleicacid sequence and a second nucleic acid duplex with a second overhangwherein the second overhang includes a second nucleic acid sequence, thefirst and second nucleic acid sequences can be at least partiallycomplementary to each other, wherein the first overhang can behybridized to a first blocking oligonucleotide over at least a portionof its length and the second overhang can be hybridized to a secondblocking oligonucleotide over at least a portion of its length, andwherein the first nucleic acid duplex can be joined to an end of a firstpolynucleotide and the second nucleic acid duplex can be joined to anend of a second polynucleotide. In some embodiments, the methods canfurther comprise the step(s): separating the first blockingoligonucleotide from the first overhang. In some embodiments, themethods can further comprise the step(s): separating the second blockingoligonucleotide from the second overhang. In some embodiments, the firstblocking oligonucleotides can be separated from the first nucleic acidoverhangs and the second blocking oligonucleotides can be separated fromthe second nucleic acid overhangs essentially simultaneously orsequentially. In some embodiments, the methods can further comprise thestep(s): hybridizing the first nucleic acid sequence with the secondnucleic acid sequence to join together the first and the secondoverhangs thereby joining the first and second nucleic acid duplexes. Insome embodiments, a first nucleic acid duplex can comprise a firstdouble-stranded nucleic acid adaptor. In some embodiments, a secondnucleic acid duplex can comprise a second double-stranded nucleic acidadaptor. In some embodiments, a first or a second double-strandednucleic acid adaptor can comprise biotin. In some embodiments, a firstpolynucleotide can be a double-stranded polynucleotide. In someembodiments, a second polynucleotide can be a double-strandedpolynucleotide. In some embodiments, a first polynucleotide can comprisea polynucleotide of interest. In some embodiments, a secondpolynucleotide can comprise a polynucleotide of interest. In someembodiments, a first and a second polynucleotide can be double-strandedpolynucleotides and can form part of a single contiguous double-strandednucleic acid molecule. In some embodiments, a first and a secondpolynucleotide can be double stranded polynucleotides and can form partof different double-stranded nucleic acid molecules.

In some embodiments, in methods for joining a first and a second nucleicacid, a first nucleic acid overhang can comprise one end of a firstpolynucleotide and a second nucleic acid overhang can comprise one endof a second polynucleotide.

In some embodiments, in methods for joining a first and a second nucleicacid, a first and a second polynucleotide can be double-strandedpolynucleotides and can form part of a single contiguous double-strandednucleic acid molecule.

In some embodiments, in methods for joining a first and a second nucleicacid, joining together the first and the second nucleic acid overhangsin step (c) can include circularizing the first and secondpolynucleotides.

In some embodiments, in methods for joining a first and a second nucleicacid, a first and a second polynucleotide can be double strandedpolynucleotides and can form part of different double-stranded nucleicacid molecules.

In some embodiments, in methods for joining a first and a second nucleicacid, the separating the first blocking oligonucleotide from the firstoverhangs can comprise denaturing the first single-stranded blockingoligonucleotide from the first overhang.

In some embodiments, in methods for joining a first and a second nucleicacid, the separating the second blocking oligonucleotide from the secondoverhangs can comprise denaturing the second single-stranded blockingoligonucleotide from the second overhang.

In some embodiments, in methods for joining a first and a second nucleicacid, methods for joining a first and a second nucleic acid can furthercomprise a step: ligating the hybridized first and the second nucleicacid sequences with a ligase enzyme.

In some embodiments, in methods for joining a first and a second nucleicacid, the first nucleic acid sequence so hybridized with the secondnucleic acid sequence to join together the first and the secondoverhangs forms a junction on each nucleic acid strand between thejoined first and the second nucleic acid overhangs.

In some embodiments, in methods for joining a first and a second nucleicacid, at least one junction can comprise a nick.

In some embodiments, in methods for joining a first and a second nucleicacid, methods for joining a first and a second nucleic acid can furthercomprise a step: moving the nick to a new position.

In some embodiments, in methods for joining a first and a second nucleicacid, a nick can be moved to a new position within the polynucleotide.

In some embodiments, in methods for joining a first and a second nucleicacid, the moving the nick to a new position within the polynucleotidecan be conducted with a nick translation reaction on the nick.

In some embodiments, in methods for joining a first and a second nucleicacid, a nick translation reaction can be a coupled 5′ to 3′ DNApolymerization/degradation reaction, or can be a coupled 5′ to 3′ DNApolymerization/strand displacement reaction.

In some embodiments, in methods for joining a first and a second nucleicacid, the length of time of a nick translation reaction can be modulatedto increase or decrease the distance to move the nick to a new positionwithin the polynucleotide of interest.

Provided herein are joined first and second nucleic acid overhangsprepared by the methods of the present teachings.

In some embodiments, methods for joining a first and a second nucleicacid can further comprise the step(s): degrading at least a portion ofthe strand having the nick, thereby opening the nick into a gap.

In some embodiments, methods for joining a first and a second nucleicacid can further comprise the step(s): cleaving the strand opposite thegap thereby releasing a linear mate pair construct.

In some embodiments, in methods for joining a first and a second nucleicacid, a degrading reaction can be performed with any exonuclease enzyme(e.g., T7 exonuclease).

In some embodiments, in methods for joining a first and a second nucleicacid, a cleaving reaction can be performed with a single-strand specificendonuclease enzyme.

Provided herein are released linear mate pair constructs prepared by themethods of the present teachings.

Provided herein is a mate pair library, comprising two or more releasedlinear mate pair constructs which are prepared by the methods of thepresent teachings.

In some embodiments, methods for joining a first and a second nucleicacid can further comprise the step of reacting the biotin withstreptavidin.

In some embodiments, methods for joining a first and a second nucleicacid can further comprise the step: joining at least one end of areleased linear mate pair construct to at least one amplificationadaptor and/or sequencing adaptor.

In some embodiments, methods for joining a first and a second nucleicacid can further comprise the step: amplifying the released linear matepair construct.

In some embodiments, methods for joining a first and a second nucleicacid can further comprise the step: sequencing the released linear matepair construct.

In some embodiments, methods for joining a first and a second nucleicacid can further comprise the step: sequencing the released linear matepair construct with a semiconductor based sequencing platform or ionsensitive sequencing platform.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1A is a schematic showing a non-limiting example of a left and aright blocking oligonucleotide adaptor used to circularize apolynucleotide of interest. (Top) A left and a right blockingoligonucleotide adaptor each comprising a duplex of a first and secondsingle-stranded oligonucleotide with an overhang portion, and a thirdsingle-stranded oligonucleotide (blocking oligonucleotide) annealed toan overhang portion. Left and right blocking adaptors can be joined to adouble-stranded polynucleotide of interest. (Bottom) A thirdsingle-stranded oligonucleotide (blocking oligonucleotide) can beremoved and the overhang portions from the left and right blockingoligonucleotide adaptors anneal with each other to circularize thepolynucleotide of interest. For the sake of clarity, the diagram showsonly a portion of the polynucleotide of interest.

FIG. 1B is a schematic showing a non-limiting example of a left and aright blocking oligonucleotide adaptor used to circularize apolynucleotide of interest. (Top) A left and a right blockingoligonucleotide adaptor each comprising a duplex of a first and secondsingle-stranded oligonucleotide with an overhang portion, and a thirdsingle-stranded oligonucleotide (blocking oligonucleotide) annealed toan overhang portion. Left and right blocking adaptors can be joined to adouble-stranded polynucleotide of interest. (Bottom) A thirdsingle-stranded oligonucleotide (blocking oligonucleotide) can beremoved and the overhang portions from the left and right blockingoligonucleotide adaptors anneal with each other to circularize thepolynucleotide of interest. For the sake of clarity, the diagram showsonly a portion of the polynucleotide of interest.

FIG. 2 is a schematic showing a non-limiting example of a left and aright blocking oligonucleotide adaptor used to circularize apolynucleotide of interest. (Top) a left and a right blockingoligonucleotide adaptor each comprising a duplex of a first and secondsingle-stranded oligonucleotide with an overhang portion, and a thirdsingle-stranded oligonucleotide (first blocking oligonucleotide)annealed to an overhang portion, and a fourth single-strandedoligonucleotide (second blocking oligonucleotide) annealed to theoverhang portion formed by the third single-stranded oligonucleotide.Left and right blocking adaptors can be joined to a double-strandedpolynucleotide of interest. (Bottom) The third and fourthsingle-stranded oligonucleotides (first and second blockingoligonucleotides) can be removed and the overhang portions from the leftand right blocking oligonucleotide adaptors anneal with each other tocircularize the polynucleotide of interest. For the sake of clarity, thediagram shows only a portion of the polynucleotide of interest.

FIG. 3 is a schematic showing a non-limiting example of a nucleotidesequence of a left and right blocking oligonucleotide adaptor, and amethod for circularizing a polynucleotide of interest which includesmoving nicks to a new position on the circular molecule with a nicktranslation reaction, and releasing a linear mate pair construct withexonuclease.

FIG. 4 is a schematic showing a non-limiting example of a method forcircularizing a polynucleotide of interest using a pair of blockingoligonucleotide adaptors and releasing a mate pair construct.

FIG. 5 is a schematic showing a non-limiting example of a method forcircularizing a polynucleotide of interest using a pair of blockingoligonucleotide adaptors and releasing a mate pair construct.

FIG. 6A is a schematic showing a non-limiting example of a blockingoligonucleotide adaptor having a hairpin structure.

FIG. 6B is a schematics showing a non-limiting example of a blockingoligonucleotide adaptor having a hairpin structure.

FIG. 7 is a schematic depicting a non-limiting example of apolynucleotide of interest joined to two different barcode adaptors(BC1, BC2) which can be joined to a left and right blockingoligonucleotide adaptor. This construct is shown prior to acircularization step.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present teachings described herein shall have themeanings that are commonly understood by those of ordinary skill in theart. Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are used,for example, for nucleic acid purification and preparation, chemicalanalysis, recombinant nucleic acid, and oligonucleotide synthesis.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications or as commonly accomplished in the artor as described herein. The techniques and procedures described hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the instant specification. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Thirded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.2000). The nomenclatures utilized in connection with, and the laboratoryprocedures and techniques described herein are those well known andcommonly used in the art.

As utilized in accordance with exemplary embodiments provided herein,the following terms, unless otherwise indicated, shall be understood tohave the following meanings:

DESCRIPTION OF VARIOUS EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as is commonly understood by one ofordinary skill in the art to which these inventions belong. All patents,patent applications, published applications, treatises and otherpublications referred to herein, both supra and infra, are incorporatedby reference in their entirety. If a definition and/or description isset forth herein that is contrary to or otherwise inconsistent with anydefinition set forth in the patents, patent applications, publishedapplications, and other publications that are herein incorporated byreference, the definition and/or description set forth herein prevailsover the definition that is incorporated by reference. It will beappreciated that there is an implied “about” prior to the temperatures,concentrations, times, etc discussed in the present teachings, such thatslight and insubstantial deviations are within the scope of the presentteachings herein. In this application, the use of the singular includesthe plural unless specifically stated otherwise. Also, the use of“comprise”, “comprises”, “comprising”, “contain”, “contains”,“containing”, “include”, “includes”, and “including” are not intended tobe limiting. As used herein, the terms “comprises,” “comprising,”“includes,” “including,” “has,” “having” or any other variation thereof,are intended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent). It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention.

Provided herein are compositions, systems, methods, and kits for joiningtogether the ends of one or more polynucleotides using at least one pairof blocking oligonucleotide adaptors. Blocking oligonucleotide adaptorscan be used to reduce the formation of adaptor dimers or trimers (orhigher-order concatemers) which can improve the yield of desirablepolynucleotide-adaptor products in any recombinant nucleic acidworkflow. A joining step can include joining together two or moredifferent polynucleotides together, or joining together two ends of onepolynucleotide to form a circular molecule.

Provided herein are blocking oligonucleotide adaptors which can be usedto join together different polynucleotides or for joining together theends of one polynucleotide. Blocking oligonucleotide adaptors can eachcomprise: (i) a nucleic acid duplex comprising a first single-strandedoligonucleotide annealed to a second single-stranded oligonucleotide toform an overhang portion; and (ii) a third single-strandedoligonucleotide (a blocking oligonucleotide) that is annealed to theoverhang portion (FIGS. 1A and B). An end of the nucleic acid duplexhaving the overhang portion can form an adaptor-joining end and theother end of the duplex can form a target-joining end. The blockingoligonucleotide (the third single-stranded oligonucleotide) can annealto the overhang portion to interfere with and/or prevent hybridizationbetween the overhang portions of the blocking oligonucleotide adaptors.Use of blocking oligonucleotide adaptors during a polynucleotide-joiningstep can reduce or prevent formation of dimers, trimers or otherconcatemerization of adaptors. Methods for joining together two or morepolynucleotides, or for joining together two ends of a polynucleotide,can be practiced on any polynucleotide(s) including DNA, cDNA, RNA,RNA/DNA hybrids, and nucleic acid analogs.

Compositions, systems, methods and kits disclosed herein can be used tojoin together the ends of one or more polynucleotides. In someembodiments, a polynucleotide-joining step can be part of anyrecombinant DNA workflow, such as preparing nucleic acid libraries forany type of analysis, including mapping or sequencing. Nucleic acidsequencing techniques, platforms, and systems for which this disclosureis useful include, among others, sequencing-by-synthesis, chemicaldegradation sequencing (e.g., Maxam-Gilbert), ligation-based sequencing,hybridization sequencing, pyrophosphate detection sequencing, capillaryelectrophoresis, gel electrophoresis, next-generation, massivelyparallel sequencing platforms, semiconductor based sequencing platforms,sequencing platforms that detect hydrogen ions or other sequencingby-products, and single molecule sequencing platforms.

Many next-generation or massively parallel sequencing systems caninvolve the preparation of nucleic acid libraries, which often includesteps for joining together two or more polynucleotides, or steps forjoining the ends of one polynucleotide to form a circular molecule. Forexample, many next-generation sequencing systems prepare and analyzemate pair libraries. In some embodiments, a mate pair can include twotags (nucleic acid sequences) that originate from one nucleic acidfragment. A mate pair library can include a collection of mate pairsthat can be used as sequencing templates. Typically, mate pair libraryworkflows can include circularizing a linear nucleic acid and releasinga linear mate pair construct. Typically, the ends of the fragments thatmake up a mate pair library can be sequenced. Generally, the distancebetween the two ends, or other information regarding the two ends, isknown. Sequence information from these ends can be aligned or mapped toa known genomic sequence for sequence assembly (Mullikin and Ning 2003Genome Res. 13:81-90; Kent 2001 Genome Res. 11:1541-1548). Mate pairlibraries are commonly used in genomic DNA physical mapping andsequencing strategies (Siegel 2000 Genomics 68:237-246; Roach 1995Genomics 26:345-353). Other types of libraries used in or fornext-generation sequencing include fragment libraries, RNA libraries(e.g., mRNA libraries, RNA-Seq libraries, whole transcriptome libraries,cell-specific RNA libraries), chromatin immunoprecipitation (ChIP)libraries, and methylated DNA libraries. Compositions, systems, methods,and kits disclosed herein can be useful for preparing nucleic acidlibraries for use with any next-generation sequencing system, including:sequencing by oligonucleotide probe ligation and detection (e.g., SOLiD™from Life Technologies, WO 2006/084131), probe-anchor ligationsequencing (e.g., Complete Genomics™ or Polonator™),sequencing-by-synthesis (e.g., Genetic Analyzer and HiSeg™, fromIllumina), pyrophosphate sequencing (e.g., Genome Sequencer FLX from 454Life Sciences), ion-sensitive sequencing (e.g., Personal Genome Machinefrom Ion Torrent™ Systems, Life Technologies), and single moleculesequencing platforms (e.g., HeliScope™ from Helicos).

Compositions, systems, methods and kits disclosed herein can be used ina workflow for constructing a nucleic acid library for sequencing in anoligonucleotide probe ligation and detection system (e.g., SOLiD™ fromLife Technologies). Provided herein are blocking oligonucleotideadaptors which can be used to join together the ends of a polynucleotideto form a circular molecule. Blocking oligonucleotide adaptors can eachcomprise: (i) a first and second single-stranded oligonucleotide thatare annealed together to form a nucleic acid duplex with an overhangportion; and (ii) a third single-stranded oligonucleotide (blockingoligonucleotide) that is annealed to the overhang portion. The end ofthe duplex structure having the overhang portion can form anadaptor-joining end and the other end of the duplex structure can form atarget-joining end. Methods for preparing a SOLiD mate-pair library cangenerally include: (a) providing a linear polynucleotide of interest(e.g., a double-stranded polynucleotide) having a first and second end;(b) joining the first end of the linear polynucleotide of interest to afirst oligonucleotide adaptor and joining the second end of the linearpolynucleotide of interest to a second oligonucleotide adaptor so as togenerate an adaptor-polynucleotide-adaptor product; and (c)circularizing the adaptor-polynucleotide-adaptor product. Acircularizing step (c) can include removing the third single-strandedoligonucleotide (blocking oligonucleotide) from the overhang portions ofthe first and second oligonucleotide adaptors so as to expose theoverhang portions of the adaptor-joining ends. The exposed overhang endscan be annealed together, thereby circularizing theadaptor-polynucleotide-adaptor product. An exposed overhang ends canhybridize to each other so as to leave a gap or nick on one or bothstrands at the junction between the adaptor-joining ends of the firstand second adaptors. The nick(s) can be moved to a new position withinthe polynucleotide of interest. For example, the nick(s) can be moved toa new position by conducting a nick translation reaction on the nick.Conducting the nick translation reaction for a shorter or longer periodof time can modulate the distance that the nick moves into thepolynucleotide of interest. A strand opposite the new position of thenick can be cleaved with a single-strand specific endonuclease enzyme torelease a linear mate pair construct having a pair of blockingoligonucleotide adaptors (minus the blocking oligonucleotide) flanked bytwo portions of the polynucleotide of interest (e.g., tags). The lengthsof the tags can be modulated by increasing or decreasing the length oftime of the nick translation reaction. The released linear mate pairconstruct can be joined to additional adaptors to permit amplificationand/or sequencing. The mate pair constructs can be sequenced usingSOLiD™ sequencing methods.

Compositions, systems, methods and kits disclosed herein can be used ina workflow for constructing a nucleic acid library for sequencing on asemiconductor based-sequencing platform. In some embodiments,compositions, systems, methods and kits disclosed herein can be used ina workflow for constructing a nucleic acid library for Ion Torrent™Systems (Life Technologies). Provided herein are blockingoligonucleotide adaptors which can be used to join together the ends ofa polynucleotide to form a circular molecule. Blocking oligonucleotideadaptors can each comprise: (i) a first and second single-strandedoligonucleotide that are annealed together to form a nucleic acid duplexwith an overhang portion; and (ii) a third single-strandedoligonucleotide (blocking oligonucleotide) that is annealed to theoverhang portion. The end of the duplex structure having the overhangportion can form an adaptor-joining end and the other end of the duplexstructure can form a target-joining end. Methods for preparing a IonTorrent™ mate-pair library can generally include: (a) providing a linearpolynucleotide of interest (e.g., a double-stranded polynucleotide)having a first and second end; (b) joining the first end of the linearpolynucleotide of interest to a first oligonucleotide adaptor andjoining the second end of the linear polynucleotide of interest to asecond oligonucleotide adaptor so as to generate anadaptor-polynucleotide-adaptor product; and (c) circularizing theadaptor-polynucleotide-adaptor product. A circularizing step (c) caninclude removing the third single-stranded oligonucleotide (blockingoligonucleotide) from the overhang portions of the first and secondoligonucleotide adaptors so as to expose the overhang portions of theadaptor-joining ends. An exposed overhang ends can be annealed together,thereby circularizing the adaptor-polynucleotide-adaptor product. Anexposed overhang ends can hybridize to each other so as to leave a gapor nick on one or both strands at the junction between theadaptor-joining ends of the first and second adaptors. A nick(s) can bemoved to a new position within the polynucleotide of interest. Forexample, the nick(s) can be moved to a new position by conducting a nicktranslation reaction on the nick. Conducting a nick translation reactionfor a shorter or longer period of time can modulate the distance thatthe nick moves into the polynucleotide of interest.

The strand opposite the new position of the nick can be cleaved with asingle-strand specific endonuclease enzyme to release a linear mate pairconstruct having a pair of blocking oligonucleotide adaptors (minus theblocking oligonucleotide) flanked by two portions of the polynucleotideof interest (e.g., tags). The lengths of the tags can be modulated byincreasing or decreasing the length of time of the nick translationreaction. The released linear mate pair construct can be joined toadditional adaptors to permit amplification and/or sequencing. The matepair constructs can be sequenced using Ion Torrent™ sequencing methods.For example, mate pair constructs can be clonally amplified on IonSphere™ Particles as part of the Ion Xpress™ Template Kit (LifeTechnologies Part No. 4469001) for use in downstream sequencing.Template preparation can be performed essentially accordingly to theprotocols provided in the Ion Xpress™ Template Kit v2.0 User Guide (LifeTechnologies, Part No. 4469004). The amplified DNA can then be sequencedon an Ion PGM™ sequencer (Ion Torrent™, Life Technologies, Part No.4462917) essentially according to the protocols provided in the IonSequencing Kit v2.0 User Guide (Ion Torrent™, Life Technologies, PartNo. 4469714) and using the reagents provided in the Ion Sequencing Kit(Ion Torrent™, Life Technologies, Part No. 4468997) and the Ion 314™Chip Kit (Ion Torrent™, Life Technologies, Part No. 4462923).

Provided herein are blocking oligonucleotide adaptors which can comprisea double-stranded oligonucleotide adaptor having: (i) a first and asecond single-stranded oligonucleotide annealed together to form aduplex having an overhang portion and (ii) a third single-strandedoligonucleotide annealed to the overhang portion, wherein the end of theduplex having the overhang portion forms an adaptor-joining end and theother end of the duplex forms a target-joining end (FIGS. 1A and B). Insome embodiments, the first single-stranded oligonucleotide can comprisea 5′ phosphorylated terminal end. In some embodiments, the secondsingle-stranded oligonucleotide can lack a 5′ phosphorylated terminalend. In some embodiments, the third single-stranded oligonucleotide canlack a 5′ phosphorylated terminal end. In some embodiments, thedouble-stranded oligonucleotide adaptor can comprise at least one biotinmoiety.

Provided herein are a circularized polynucleotide of interest which cancomprise: a linear polynucleotide of interest having a first end and asecond end, wherein the first end can be joined to a target-joining endof a first double-stranded oligonucleotide adaptor and the second endcan be joined to a target-joining end of a second double-strandedoligonucleotide adaptor, and having the third single-strandedoligonucleotides removed from the first and second double-strandedoligonucleotide adaptors so as to anneal together the overhang portionsof the first and second double-stranded oligonucleotide adaptors therebyforming a circular polynucleotide of interest, wherein the first andsecond double-stranded oligonucleotide adaptors include (i) a first anda second single-stranded oligonucleotide annealed together to form aduplex having an overhang portion and (ii) a third single-strandedoligonucleotide annealed to the overhang portion, wherein the end of theduplex having the overhang portion forms a adaptor-joining end and theother end of the duplex forms a target-joining end, and wherein theoverhang portion of the first and second double-stranded oligonucleotideadaptors are capable of annealing with each other (FIGS. 1A and B). Insome embodiments, the first single-stranded oligonucleotide can comprisea 5′ phosphorylated terminal end. In some embodiments, the secondsingle-stranded oligonucleotide can lack a 5′ phosphorylated terminalend. In some embodiments, the third single-stranded oligonucleotide canlack a 5′ phosphorylated terminal end. In some embodiments, thedouble-stranded oligonucleotide adaptor can comprise a biotin moiety. Insome embodiments, in the circularized polynucleotide of interest, thejunctions between the annealed overhang portions of the first and thesecond double-stranded oligonucleotide adaptors include a gap or nick.In some embodiments, the linear polynucleotide of interest comprises adouble-stranded nucleic acid.

Provided herein are methods for circularizing nucleic acids which cancomprise: (a) providing a first and a second double-strandedoligonucleotide adaptor each having (i) a first and a secondsingle-stranded oligonucleotide annealed together to form a duplexhaving an overhang portion and (ii) a third single-strandedoligonucleotide annealed to the overhang portion, wherein the overhangportion of the first and second double-stranded oligonucleotide adaptorsare capable of annealing with each other, and wherein for the first anda second double-stranded oligonucleotide adaptors the end of the duplexhaving the overhang portion forms an adaptor-joining end and the otherend of the duplex forms a target-joining end; (b) joining thetarget-joining end of the first double-stranded oligonucleotide adaptorto a first end of the linear double-stranded polynucleotide of interest;(c) joining the target-joining end of the second double-strandedoligonucleotide adaptor to a second end of the linear double-strandedpolynucleotide of interest; (d) removing the third single-strandedoligonucleotide from the first and the second double-strandedoligonucleotide adaptors so as to expose the overhang portions of theadaptor-joining ends; and (e) annealing the overhang portions of theadaptor-joining ends thereby forming a circular polynucleotide ofinterest. In some embodiments, the junction between the adaptor-joiningends of the first and the second double-stranded oligonucleotideadaptors which are annealed together in step (e) can include at leastone nick. In some embodiments, the method can further comprise the step:moving the at least one nick to a new position within thedouble-stranded polynucleotide of interest. In some embodiments, themoving the at least one nick to a new position within thedouble-stranded polynucleotide of interest can comprise performing anick translation reaction on the at least one nick. In some embodiments,the nick translation reaction can be a coupled 5′ to 3′ DNApolymerization/degradation reaction, or can be a coupled 5′ to 3′ DNApolymerization/strand displacement reaction. In some embodiments, themethod can further comprise the steps: (f) performing an exonucleasereaction to remove at least a portion of the strand having the nick soas to open the nick into a gap; and (g) cleaving the strand opposite thegap so as to release a linear mate pair construct.

Provided herein are blocking oligonucleotide adaptors that can be usedas part of a workflow for joining together the two ends of apolynucleotide to form a circular molecule. In some embodiments,workflows can be used to prepare mate pair libraries for any nextgeneration sequencing platform.

Provided herein are workflows for preparing a next generation sequencinglibrary which can generally include: fragmenting, adaptor-joining,circularizing, and releasing a linear mate pair construct. For example,preparing a next generation mate pair library can include: (a) joiningboth ends of a polynucleotide of interest to a pair of blockingoligonucleotide adaptors; and (b) circularizing the polynucleotide ofinterest by removing the blocking oligonucleotide to permithybridization of the overhang ends and to form at least one nick at thejunction of the annealed overhang ends. In some embodiments, methodsfurther comprise: (c) moving the at least one nick to a new positionwithin the polynucleotide of interest; and (d) cleaving strand oppositethe new position of the nick so as to release a linear mate pairconstruct. In some embodiments, released mate pair constructs can bejoined to additional adaptors to form adaptor-mate pair constructs whichcan be compatible with any next generation sequencing platform. In someembodiments, additional adaptors can provide functionality foramplification, attachment to a surface and/or sequencing. In someembodiments, adaptor-mate pair constructs can be immobilized to asurface. In some embodiments, any reaction for preparing a nextgeneration sequencing library can be conducted in a reaction vessel. Insome embodiments, any reaction for preparing a next generationsequencing library can be conducted in a thermal-control apparatus.

For example, blocking oligonucleotide adaptors can generally include anucleic acid duplex having an overhang end, and a third oligonucleotide(blocking oligonucleotide) can be annealed to the overhang end. One orboth strands in the nucleic acid duplex can comprise at least onebinding partner (e.g., biotin). The end of the duplex having theoverhang end can form an adaptor-joining end and the other end of theduplex structure can form a target-joining end. In a pair of blockingoligonucleotide adaptors, the adaptor-joining ends can anneal with eachother upon removal of the third polynucleotides (blockingoligonucleotides). A 5′ terminal end of a target-joining end can bephosphorylated or can lack phosphorylation. A 5′ terminal end of anadaptor-joining end can be phosphorylated or can lack phosphorylation.

In some embodiments, a polynucleotide of interest can be subjected to anucleic acid workflow for nucleic acid library preparation that includesa fragmentation step followed by any combination and in any order anynucleic acid manipulation step, including: size selection, end repair,adaptor ligation or oligonucleotide ligation (e.g., blockingoligonucleotide adaptors, barcodes adaptors, or P1, P2, A or CAPadaptors), adaptor annealing, circularization, moving-a-nick, releasinga linear mate pair construct, nick translation, tailing, amplification,purification, removing linear nucleic acids, washing, quantization,immobilization, denaturation. In some embodiments, additional nucleicacid manipulation steps can include: exonuclease or endonucleasereactions. In some embodiments, any step or any combination of steps canbe conducted by an automated system. For example, an automated systemcan include robotic delivery of reagents and/or computer-controlledreaction regimen (temperature and time). Any of these steps can beomitted or repeated. For example generating a mate pair library for a 3kb insert can include: fragmenting the polynucleotide of interest,size-selection, end repair, adaptor ligation or oligonucleotideligation, and circularization. In some embodiments, generating a matepair library for a 10 kb insert can include: fragmenting thepolynucleotide of interest, end repair, size-selection, adaptor ligationor oligonucleotide ligation, and circularization.

For example, a nucleic acid library preparation workflow can include thesteps: fragmentation, size-selection; end repair; ligation to blockingoligonucleotide adaptors; circularization; nick translation; releasinglinear mate pair constructs; tailing; ligation to sequencing adaptors;nick translation and amplification.

In another example, a nucleic acid library preparation workflow caninclude the steps: fragmentation, end repair; size-selection; ligationto blocking oligonucleotide adaptors; circularization; nick translation;releasing linear mate pair constructs; tailing; ligation to sequencingadaptors; nick translation and amplification.

In another example, a nucleic acid library preparation workflow caninclude the steps: fragmentation, end repair; size-selection; ligationto blocking oligonucleotide adaptors; circularization; nick translation;releasing linear mate pair constructs; end repair; ligation tosequencing adaptors; amplification; and size-selection.

In another example, a nucleic acid library preparation workflow caninclude the steps: fragmentation; end repair; size-selection;circularization (without circularization adaptor); fragmentation ofcircularized constructs; end repair; tailing; ligation to sequencingadaptors; amplification; size-selection; and immobilization.

In another example, a nucleic acid library preparation workflow caninclude the steps: fragmentation; end repair; ligation tocircularization adaptors (e.g., loxP adaptors); size-selection; fill-inreaction; circularization (e.g., via Cre recombinase); fragmentation ofcircularized constructs; end repair; ligation to sequencing adaptors;amplification; size-selection; immobilization; and nucleic aciddenaturation.

In some embodiments, a polynucleotide of interest can be isolated fromany source including: an organism; normal or diseased cells or tissues;fresh or archived (e.g., formalin and/or paraffin) cell or tissuesamples; chromosomal, genomic, organellar, methylated, cloned,amplified, DNA, cDNA, RNA, RNA/DNA or synthesized.

In some embodiments, a polynucleotide of interested can be fragmented bymechanical stress, enzymatic or chemical methods. Mechanical stressincludes sonication, nebulization or cavitation. Enzymatic fragmentationincludes any restriction endonuclease, nicking endonuclease orexonuclease. Chemical fragmentation includes dimethyl sulfate,hydrazine, NaCl, piperidine, or acid. In some embodiments,polynucleotides of interest can be fragmented to yield fragments thatare about 3 kb or about 10 kb in length.

In some embodiments, fragmented polynucleotides of interest can besubjected to any size-selection procedure to obtain any desired sizerange. In some embodiments, nucleic acid size selection method includeswithout limitation: solid phase adherence or immobilization;electrophoresis, such as gel electrophoresis; and chromatography, suchas HPLC and size exclusion chromatography. In some embodiments,size-selected fragments can include about 0.9-1.3 kb, or about 0.8-1.4kb, or about 1.5-6 kb, or about, or about 2-5 kb, or about 2.8-3.5 kb,or about 6.5-9.5 kb, or about 10-11 kb, or about 17-25 kb. In someembodiments, fragmented polynucleotides of interest can be size selectedfrom a 1% or a 0.6% agarose gel.

In some embodiments, the ends and/or internal portions of a fragmentedpolynucleotide of interest can be repaired to remove 5′ and/or 3′overhang ends, or to phosphorylate an end or remove a terminal phosphategroup. For example, an overhang end can be filled-in with a polymerase,such as a DNA polymerase (e.g., T4 DNA polymerase or Bst DNA polymerase)or a Klenow (e.g., large fragment and/or exonuclease minus). In someembodiments, an overhang end can be filled-in with natural nucleotidesor analogs, including biotinylated nucleotides. In some embodiments,terminal 5′ phosphate groups can be removed with a kinase enzyme. Insome embodiments, an end repair reaction can include adding a phosphateto a 5′ end and/or removing a phosphate from a 3′ end. These reactionscan be conducted with one or more enzymes that catalyze addition of aphosphate group to a 5′ terminus of a single-stranded or double-strandednucleic acid and/or that catalyze removal of 3′ phosphoryl groups from anucleic acid. In some embodiments, addition or removal of a phosphategroup can be catalyzed by a polynucleotide kinase. A polynucleotidekinase can be a T4 polynucleotide kinase, or can be isolated from othersources (e.g., human). A polynucleotide kinase reaction can be conductedin the presence of ATP. In some embodiments, an end repair reactionresulting in a phosphate to a 5′ end catalyzed by one or more enzymescan be terminated by a heat inactivation step.

In some embodiments, a polynucleotide of interest can have a first endand a second end. In some embodiments, a first end can be joined to thetarget-joining end of a first blocking oligonucleotide adaptor. In someembodiments, a second end can be joined to the target-joining end of asecond blocking oligonucleotide adaptor. In some embodiments, one ormore blocking oligonucleotide adaptors can be joined to a polynucleotideof interest enzymatically or by annealing. In some embodiments,adaptor-joining can be conducted with a ligase enzyme. In someembodiments, each end of a polynucleotide of interest can be joined to aseparate blocking oligonucleotide adaptor to form anadaptor-polynucleotide-adaptor product.

In some embodiments, polynucleotides of interest can be subjected to anypurification procedure to remove non-desirable materials. In someembodiments, purification procedures can include: bead purification,column purification, gel electrophoresis, dialysis, alcoholprecipitation, and size-selective PEG precipitation. For example, apurification step can be conducted with solid phaseadherence/immobilization paramagnetic beads (AMPure XP beads fromAgencourt) or streptavidin paramagnetic beads (Dynabeads™ fromInvitrogen). In another example, PureLink™ (Invitrogen) or Microcon™(Millipore) columns or can be used for purification.

In some embodiments, an adaptor-polynucleotide-adaptor product can becircularized by removing the blocking oligonucleotides (thirdoligonucleotides) to permit annealing between the overhang ends(adaptor-joining ends) of the two adaptors thereby circularizing theadaptor-polynucleotide-adaptor product. In some embodiments, a blockingoligonucleotide (e.g., third oligonucleotide) can be separated(denatured) from an overhang portion by heat denaturation and/ormodulating the salt and/or sodium and/or formamide concentration(s). Insome embodiments, a blocking oligonucleotide can be removed, and a firstand a second overhang end can anneal with each other at about 30° C., orabout 50-80° C. or about 70° C. In some embodiments, a polynucleotide ofinterest can undergo intramolecular circularization (via ligation orannealing) without joining to a circularization adaptor (e.g.,self-circularization). Circularization (without a circularizationadaptor) can be achieved with a ligase at about 4-35° C. In someembodiments, a polynucleotide of interest can be joined to a loxPadaptor and circularization can be mediated by a Cre recombinase enzymereaction. Circularization with Cre recombinase can be achieved at about4-35° C.

In some embodiments, in a circularized construct, the junctions betweenthe annealed overhang ends can include at least one nick or gap. Nicksor gaps can serve as an initiation site for an enzymatic reaction tomove the position of the nick or gap to a new position.

In some embodiments, the position of the nick or gap at the junctionbetween the annealed overhang ends can be moved to a new position withinthe polynucleotide of interest. For example, a nick translation reactioncan be conducted for a period of time to move the position of the nickto a new position within the polynucleotide of interest. The nicktranslation reaction can be stopped at a desired time. The length oftime for conducting the nick translation reaction can providesize-tunable tags in the released mate pair construct. In someembodiments, a nick translation reaction can be conducted with an enzymethat couples a 5′→3′ polymerization/degradation reaction, such as E.coli DNA polymerase I or Bst DNA polymerase.

In some embodiments, after conducting a nick translation reaction, alinear mate pair construct can be released from a circular molecule bycleaving the strand opposite the new position of the nick. A cleavingstep can be conducted with an enzyme or chemical compound. For example,cleavage can be conducted with one or more endonucleases and/orexonucleases together or serially. In some embodiments, releasing alinear mate pair construct can include the steps: enzymaticallyopening/widening the nick (at the new position) and cleaving the strandopposite the widened nick site. In some embodiments, a T7 exonucleasecan be used to widen the nick, and an S1 nuclease can be used to cleavethe strand opposite the nick. In some embodiments, a released linearmate pair construct can include a pair of blocking oligonucleotideadaptors (joined together) flanked on both sides by pairedpolynucleotide sequences of interest (e.g., left and right paired tags).In some embodiments the polynucleotide sequence of interest (e.g., leftor right paired tag) can be about 25 to about 1000 base pairs, about 25to about 500 base pairs, about 25 to about 300 base pairs, about 50 toabout 200 base pairs, or about 50 to about 100 base pairs in length.

In some embodiments, a non-template-dependent terminal transferasereaction can be conducted for a tailing step. In some embodiments, anon-template-dependent terminal transferase reaction can be catalyzed bya Taq polymerase, Tfi DNA polymerase, 3′ exonuclease minus-large(Klenow) fragment, or 3′ exonuclease minus-T4 polymerase.

In some embodiments, one or both ends of a released mate pair constructcan be joined to at least one additional adaptor. Such additionaladaptors can include functionalities for further nucleic acidmanipulations such as amplification, immobilization, sequencing and/orunique identification. In some embodiments, each end of a released matepair construct can be joined to the same or different additionaladaptors. In some embodiments, a released mate pair construct can bejoined to at least one additional adaptor with a ligase enzyme or byhybridization. In some embodiments, additional adaptors can have anystructure, including linear, hairpin, forked, or stem-loop. A releasedmate pair construct can be joined to an adaptor to permit attachment toa particle (e.g., bead) or to a surface. For example, an adaptor caninclude a nucleotide sequence that is complementary to anoligonucleotide capture primer that is attached to a particle orsurface. An immobilized oligonucleotide capture primer can anneal to animmobilization adaptor that is joined to a released mate pair construct,and a primer extension reaction can be conducted to generate acomplementary copy of the released mate pair construct attached to asurface. In some embodiments, a bridge amplification reaction can beconducted by joining a released mate pair construct to differentadaptors at each end (where the adaptors are complementary to differentoligonucleotide capture primers that are attached to a surface) andconducting multiple primer extension reactions. In some embodiments,attachment of a released mate pair construct to a particle or surfacecan be achieved by conducting a primer extension reaction or anamplification reaction in an aqueous condition. Primer extension andamplification reactions can be conducted under isothermal orthermocyclic conditions, or can be reacted in a tube, a well, anoil-and-water emulsion droplet or an agarose droplet (Yang 2010 Lab Chip10(21):2841-2843).

In some embodiments, a released mate pair construct can be amplifiedusing at least one type of polymerase, nucleotides (natural or analogsthereof), and one or more amplification primers (e.g., forward and/orreverse primers). In some embodiments, the linear mate pair can beamplified by emulsion polymerase chain reaction (EmPCR). In someembodiments, multiple amplification cycles can be conducted. Forexample, a limited number of amplification cycles can be conducted(e.g., 10-14, 12-16, 16-20 or 20 cycles or more), or amplification canbe conducted until product plateau is achieved. In some embodiments,amplification can be conducted with a thermostable or thermo-labilepolymerase. In some embodiments, amplification can be conducted with apolymerase having proofreading capability. In some embodiments,amplification can be conducted with a Taq polymerase, Phusion™polymerase (Finnzyme, Finland), a GC-rich DNA polymerase such as oneisolated from Pyrolobus fumarius (e.g., AccuPrime™ from Invitrogen,Carlsbad, Calif.), or a blend of different DNA polymerases foramplifying GC-rich sequences (e.g., GC-rich PCR system from Roche).

In some embodiments, one or both ends of a released mate pair constructcan be modified for attachment to a surface or particle. For example, a5′ or 3′ end can be modified to include an amino group that can bind toa carboxylic acid compound on a surface or a particle. A 5′ end caninclude a phosphate group for reacting with an amine-coated surface (orparticle) in the presence of a carbodiimide (e.g., water solublecarbodiimide). A nucleic acid can be biotinylated at one end to bindwith an avidin-like compound (e.g. streptavidin) attached to a surface.

In some embodiments, a surface can be planar, convex, concave, or anycombination thereof. A surface can be porous, semi-porous or non-porous.A surface can comprise an inorganic material, natural polymers,synthetic polymers, or non-polymeric material. A surface includes aflowcell, well, groove, channel, reservoir, filter, gel or inner wallsof a capillary. A surface can be coated with an acrylamide compound. Amate pair construct can be immobilized to an acrylamide compound coatingon a surface.

In some embodiments, a mate pair construct can be attached to aparticle. In some embodiments, a particle can have a shape that isspherical, hemispherical, cylindrical, barrel-shaped, toroidal,rod-like, disc-like, conical, triangular, cubical, polygonal, tubular,wire-like or irregular. A particle can have an iron core or comprise ahydrogel or agarose (e.g., Sepharose™). A particle can be paramagnetic.A particle can be spherical or irregular shape. A particle can havecavitation or pores, or can include three-dimensional scaffolds. Aparticle can be coated with a carboxylic acid compound or an aminecompound for attaching nucleic acid fragments. A particle can be coatedwith an avidin-like compound (e.g., streptavidin) for bindingbiotinylated nucleic acid fragments. Particles can be deposited to asurface of a sequencing instrument. Sequencing reagents can be deliveredto the deposited particles to conduct sequencing reactions. In someembodiments, a mate pair construct can be attached or immobilized to IonSphere™ Particles (sold as a component of the Ion Xpress Template Kit(Part No. 4469001)) for clonal amplification and use in downstreamsequencing Immobilization to Ion Sphere™ Particles can be performedessentially according to the protocols provided in the Ion Xpress™Template Kit v2.0 User Guide (Part No.: 4469004)).

In some embodiments, any step for preparing a next generation librarycan be conducted in any type of reaction vessel. For example, a reactionvessel includes any type of tube, column or well (e.g., 96-well plate).

In some embodiments, any step for preparing a next generation library(e.g., a mate pair library) can be practiced in any type ofthermal-control apparatus. In some embodiments, a thermal-controlapparatus can maintain a desired temperature, or can elevate anddecrease the temperature, or can elevate and decrease the temperaturefor multiple cycles. In some embodiments, a thermal-control apparatuscan maintain a temperature range of about 0° C.-100° C., or can cyclebetween different temperature ranges of about 0° C.-100° C. Examples ofthermal-control apparatus include: a water bath and thermal cyclermachine. Many thermal cycler machines are commercially-available,including (but not limited to) Applied Biosystems, Agilent, Eppendorf,Bio-Rad and Bibby Scientific.

Blocking Oligonucleotide Adaptors

Provided herein are blocking oligonucleotide adaptors for joiningtogether the ends of one or more polynucleotides. A joining step caninclude joining together two or more different polynucleotides together,or joining together two ends of one polynucleotide to form a circularmolecule. Blocking oligonucleotide adaptors can be used in a recombinantnucleic acid workflow to reduce the formation of adaptor dimers ortrimers (or higher-order concatemers) which can improve the yield ofdesirable polynucleotide-adaptor products.

In some embodiments, a blocking oligonucleotide adaptor can comprise oneor more single-stranded oligonucleotides (e.g., a first and secondsingle-stranded oligonucleotide) which can anneal to each other to forma duplex structure having an overhang portion. The duplex structure canhave a 5′ or 3′ overhang portion (FIGS. 1A and B). A thirdsingle-stranded oligonucleotide (or a single-stranded portion of anoligonucleotide) can anneal to the overhang portion. In someembodiments, the end of the duplex structure having the overhang portioncan form an adaptor-joining end and the other end of the duplexstructure can form a target-joining end (FIGS. 1-5). The term“oligonucleotide”, refers to polydeoxyribonucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose) and toany polynucleotide, which can be a ribo sugar-phosphate backboneconsisting of an N-glycoside of a purine or pyrimidine base, or modifiedpurine or pyrimidine base. There is no intended distinction between thelength of a “nucleic acid”, “polynucleotide” or an “oligonucleotide”.

In some embodiments, a first and/or second single-strandedoligonucleotide can include a binding partner moiety (e.g., biotin). Insome embodiments, the first single-stranded oligonucleotide can comprisea 5′ phosphorylated terminal end. In some embodiments, the secondsingle-stranded oligonucleotide can lack a 5′ phosphorylated terminalend. In some embodiments, the third single-stranded oligonucleotide canlack a 5′ phosphorylated terminal end.

In some embodiments, a blocking oligonucleotide adaptor can comprise (i)a first single-stranded oligonucleotide annealed to a secondsingle-stranded oligonucleotide to form a duplex having an overhangportion and (ii) a third single-stranded oligonucleotide (blockingoligonucleotide) annealed to the overhang portion, where the end of theduplex having the overhang portion forms a adaptor-joining end and theother end of the duplex forms a target-joining end. The overhang portioncan be exposed by removal of the third single-stranded oligonucleotide.An exposed overhang portion of a first blocking oligonucleotide adaptorcan anneal to an exposed overhang portion of a second blockingoligonucleotide adaptor thereby joining the two adaptors together (FIGS.1-5). In some embodiments, a first and/or second single-strandedoligonucleotide can include a binding partner moiety (e.g., biotin).

Linear and Circular Polynucleotide Constructs

Provided herein are linear polynucleotide constructs, comprising alinear polynucleotide of interest joined at one or both ends with ablocking oligonucleotide adaptor.

For example, a polynucleotide of interest having a first end and asecond end can be joined at the first end to a target-joining end of afirst blocking oligonucleotide adaptor. In some embodiments, apolynucleotide of interest (which is joined to a first blockingoligonucleotide adaptor) can further include a second end joined to atarget-joining end of a second blocking oligonucleotide adaptor. In someembodiments, a linear construct comprises a polynucleotide of interestflanked on one or both sides with a blocking oligonucleotide adaptor.

In some embodiments, the overhang portion of the first and secondblocking oligonucleotide adaptors are capable of annealing with eachother. In some embodiments, the third single-stranded oligonucleotide(blocking oligonucleotide) of the first and second blockingoligonucleotide adaptors can be removed to expose the overhang portions.In some embodiments, the overhang portions of the first and secondblocking oligonucleotide adaptors can anneal with each other therebyjoining together the first and second adaptors (at their adaptor-joiningends) so as to circularize the polynucleotide of interest. In someembodiments, one or both polynucleotide strands at the junction betweenthe adaptor-joining ends of the first and second adaptors can include anick or gap.

Embodiments of Blocking Oligonucleotide Adaptors

In some embodiments, blocking oligonucleotide adaptors comprise at leastone single-stranded oligonucleotide(s) (strand(s)) (FIGS. 1 and 2). Forexample, a first and a second single-stranded oligonucleotide can annealto each other to form a double-stranded adaptor (a duplex) having a 3′or 5′ overhang portion. A third single-stranded oligonucleotide cananneal to an overhang portion. The third oligonucleotide can be ablocking oligonucleotide. One end of the double-stranded adaptor can bea target-joining end that can be joined to the polynucleotide ofinterest. The other end of the double-stranded adaptor can be anadaptor-joining end that can be joined to another adaptor (e.g., anotherblocking oligonucleotide adaptor) (FIGS. 1 and 2).

In some embodiments, a blocking oligonucleotide adaptor can have morethan one blocking oligonucleotide. For example, a third single-strandedoligonucleotide can be a first blocking oligonucleotide that anneals toan overhang portion. A third single-stranded oligonucleotide can form anoverhang portion. A fourth single-stranded oligonucleotide can be asecond blocking oligonucleotide that can anneal to an overhang formed bythe third single-stranded oligonucleotide (FIG. 2). In some embodiments,a fourth single-stranded oligonucleotide can form an overhang portion.It will become readily apparent to the skilled artisan that multipleblocking oligonucleotides can be formed from additional single-strandedoligonucleotides. Thus, the overhang portion(s) can be formed by thefirst or second single-stranded oligonucleotide or can be formed by anyof the blocking oligonucleotides. A blocking oligonucleotide (e.g.,third or fourth single-stranded oligonucleotide) can interfere withand/or prevent undesirable hybridization of the overhang portion toanother nucleic acid, such as an overhang portion from another blockingoligonucleotide adaptor or a polynucleotide of interest.

In some embodiments, a first portion of the first single-strandedoligonucleotide can anneal with a second single-strandedoligonucleotide, and a second portion of the first single-strandedoligonucleotide can form a hairpin structure which can blockhybridization of the first single-stranded oligonucleotide with anothernucleic acid (FIG. 6A). In some embodiments, a portion of the third (orfourth) single-stranded oligonucleotide can form a hairpin structurewhich can block hybridization of the third (or fourth) single-strandedoligonucleotide with another nucleic acid (FIG. 6B).

In some embodiments, the blocking oligonucleotide adaptors can beprepared by annealing together a first and second single-strandedoligonucleotide under conditions suitable for nucleic acid hybridizationto form a nucleic acid duplex having an overhang portion. In someembodiments, the overhang portion of the nucleic acid duplex can beannealed with a third single-stranded oligonucleotide under conditionssuitable for nucleic acid hybridization to form a blockingoligonucleotide adaptor (FIGS. 1 and 2, left or right adaptors).

In some embodiments, the first, second, third, fourth (and other)single-stranded oligonucleotides can hybridize (e.g., anneal) underconditions suitable for increasing or decreasing the stability of theannealed oligonucleotides. Such conditions can include salts (e.g.,sodium), temperature, pH, buffers, formamide, and the like.

In some embodiments, the blocking oligonucleotide adaptors, comprisingthe first, second, third, and optionally fourth (or more)single-stranded oligonucleotides, can be pre-assembled (annealed orhybridized) prior to joining to the polynucleotide of interest.

In some embodiments, a duplex formed by annealing a first and secondsingle-stranded oligonucleotide can be joined to a polynucleotide ofinterest, and a blocking oligonucleotide can be annealed to an overhangportion after the joining step.

In some embodiments, the first and/or second single-strandedoligonucleotides and/or any of the blocking oligonucleotides can includedeoxyribonucleotides (e.g., DNA) or ribonucleotides (e.g., RNA) or canbe a DNA/RNA hybrid.

In some embodiments, first and/or second single-strandedoligonucleotides and/or any of the blocking oligonucleotides can be anylength, including about 2-25 bases, or about 25-50 bases, or about 50-75bases, or about 75-100 bases, or about 100-150 bases, or about 150-200bases, or longer. In some embodiments, first and/or secondsingle-stranded oligonucleotides and/or any of the blockingoligonucleotides can about 0.2-1 kb, or about 1-100 kb, or about 0.1-100mega bases, or longer.

In some embodiments, any of the overhang portions formed by the first orsecond single-stranded oligonucleotide or formed by any of the blockingoligonucleotides can be any length, such as for example, about 1-20bases, or about 20-40 bases, or about 40-60 bases, or about 60-80 bases,or about 80-100 bases, or about 100-200 bases, or longer. In someembodiments, any of the overhang portions can about 0.2-1 kb, or about1-100 kb, or about 0.1-100 mega bases, or longer.

In some embodiments, the blocking oligonucleotide can be the samelength, or shorter or longer than the overhang portion to which itanneals. In some embodiments, the blocking oligonucleotide can havingsame number of bases, or fewer or more bases compared to the overhangportion to which it anneals.

In some embodiments, the first and/or second single-strandedoligonucleotides and/or any of the blocking oligonucleotides can haveany nucleotide sequence. For example, the sequence can be monomeric(e.g., TTTT or GGGG), polymeric, palindrome, non-palindrome, repetitive,or any other type of sequence.

In some embodiments, the first and/or second single-strandedoligonucleotides and/or any of the blocking oligonucleotides can includenatural or analogs of nucleosides: adenosine, thymidine, cytosine,guanine, uridine or inosine (or analogs thereof).

In some embodiments, the members of a pair of blocking oligonucleotideadaptors (e.g., left and right adaptors), can have the same or differentsequences.

In some embodiments, the first and/or second single-strandedoligonucleotides and/or any of the blocking oligonucleotides can haveany percent GC content.

In some embodiments, the portion of the first and second single-strandedoligonucleotides that anneal to each other can be partially or whollycomplementary to each other.

In some embodiments, the overhang portion and the blockingoligonucleotide that anneals to the overhang portion can be partially orwholly complementary to each other.

A nucleic acid strand that is “complementary” refers to a nucleic acidsequence-strand or a peptide nucleic acid sequence strand which whenaligned with the nucleic acid sequence of one strand of the targetnucleic acid, such that the 5′ end of the sequence is paired with the 3′end of the other sequence in antiparallel association, a stable duplexis formed. Complementarity need not be perfect. Stable duplexes can beformed that include mismatched nucleotides. Complementary nucleic acidstrands need not hybridize with each other across their entire length.

In some embodiments, a blocking oligonucleotide can be hybridized to anoverhang portion and then separated (e.g., denatured) from the overhangportion. For example, a blocking oligonucleotide can be denatured froman overhang portion under conditions that are suitable for decreasingthe stability of the annealed oligonucleotides thereby causing partialor complete denaturation. Parameters for decreasing the stability ofhybridized nucleic acids can be predicted from the length, % GC content,and/or degree of complementarity of the nucleic acid(s) to be hybridizedor denatured. In some embodiments, thermal melting temperature (T_(m))for nucleic acids can be a temperature at which half of the nucleic acidstrands are double-stranded and half are single-stranded under a definedcondition. In some embodiments, a defined condition can include ionicstrength and pH in an aqueous reaction condition. A defined conditioncan be modulated by altering the concentration of salts (e.g., sodium),temperature, pH, buffers, and/or formamide. Typically, the calculatedthermal melting temperature can be considered to be a very stringenthybridization condition under which the nucleic acids remain hybridized.A less stringent hybridization condition can be at about 5-30° C. belowthe T_(m), or about 5-25° C. below the T_(m), or about 5-20° C. belowthe T_(m), or about 5-15° C. below the T_(m), or about 5-10° C. belowthe T_(m). Methods for calculating a T_(m) are well known and can befound in Sambrook (1989 in “Molecular Cloning: A Laboratory Manual”,2^(nd) edition, volumes 1-3). Other sources for calculating a T_(m) forhybridizing or denaturing nucleic acids include OligoAnalyze (fromIntegrated DNA Technologies) and Primer3 (distributed by the WhiteheadInstitute for Biomedical Research). In some embodiments, two nucleicacids can denature from each other (e.g., blocking oligonucleotide andoverhang portion) at about 35-40° C., or about 40-45° C., or about45-50° C., or about 50-55° C., or about 55-60° C., or about 60-65° C.,or about 65-70° C., or about 70-75° C., or about 75-80° C., or about80-85° C., or 85-90° C., or higher temperature ranges.

In some embodiments, a duplex having first oligonucleotide hybridized toa second oligonucleotide can be more stable (e.g., have a higher meltingpoint) compared to a duplex having an overhang hybridized to a blockingoligonucleotide. In some embodiments, in a first blockingoligonucleotide adaptor, a first duplex comprises a firstoligonucleotide hybridized to a second oligonucleotide, where one end ofthe first oligonucleotide extends beyond the area of hybridization toform a first overhang. In some embodiments, in a second blockingoligonucleotide adaptor, a second duplex comprises a thirdoligonucleotide hybridized to a fourth oligonucleotide, where one end ofthe third oligonucleotide extends beyond the area of hybridization toform a second overhang. In some embodiments, a first blockingoligonucleotide can be hybridized to the first overhang to form a thirdduplex. In some embodiments, a second blocking oligonucleotide can behybridized to the second overhang to form a fourth duplex. In someembodiments, the melting points of the first and second duplexes can begreater than (e.g., a range of slightly through substantially greaterthan) the melting points of the third and fourth duplexes. In someembodiments, conditions used to denature (separate) the first and secondblocking oligonucleotides from their respective overhangs does notdestabilized hybridization (or does not substantially destabilizehybridization) between the first and second oligonucleotides or betweenthe third and fourth oligonucleotides.

In some embodiments, the first and/or second single-strandedoligonucleotides and/or any of the blocking oligonucleotides can includenatural nucleotides and/or nucleotide analogs. For example, first and/orsecond single-stranded oligonucleotides and/or any of the blockingoligonucleotides can include an nucleotide analog that can increase ordecrease the stability of the annealed first, second, third or fourthsingle-stranded oligonucleotides (other additional single-strandedoligonucleotides) using any combination of PNA, L-DNA, LNA (lockednucleic acids), iso-C/iso-G, L-RNA, and/or O-methyl RNA.

In some embodiments, the 5′ end of the first or second single-strandedoligonucleotide includes or lacks a terminal phosphate group.

In some embodiments, the 3′ end of the first or second single-strandedoligonucleotide includes or lacks a nucleoside tail of one or morenucleosides (e.g., a tail comprising A, G, C, T, U and/or I).

In some embodiments, a nick can be located at or near the overhangportions that are annealed together (FIGS. 1-3). In some embodiments,the number and location of the nick(s) can be adjusted by varying thelength and/or number of the overhang portions and/or by varying thelength and/or number of blocking oligonucleotides. In some embodiments,a nick can be located on different strands. In some embodiments, a firstor second single-stranded oligonucleotide can include at least one nick.

In some embodiments, the target-joining end can have a blunt end, a 3′overhang end, or a 5′ overhang end.

In some embodiments, an adaptor-joining end can include a feature thatreduces adaptor annealing. For example, in a pair of blockingoligonucleotide adaptors, an adaptor-joining end can include one or moreterminal non-complementary bases (e.g., terminal T shown in FIGS. 2 and4). In some embodiments, a 5′ end of the third single-strandedoligonucleotide or a 3′ end of the fourth single-strandedoligonucleotide can include terminal non-complementary base(s) (FIGS. 2and 4). In some embodiments, a 5′ end of the second oligonucleotide caninclude or lack a terminal 5′ phosphate group (FIGS. 1-5). One skilledin the art will readily recognize that other embodiments are possiblefor blocking oligonucleotide adaptors having single-strandedoligonucleotides forming a 5′ overhang end.

In some embodiments, a blocking oligonucleotide adaptor can include oneor more barcode sequence. For example, any of the oligonucleotides thatmake up the blocking oligonucleotide adaptors can include one or morebarcode sequences (FIG. 7). In some embodiments, a blockingoligonucleotide adaptor can be joined to one or more barcoded nucleicacid (e.g., barcoded adaptor). A barcode sequence can be a uniqueidentifying sequence. A barcode sequence can be used for identifying,sorting, tracking, capture or multiplex reactions.

In some embodiments, any of the oligonucleotides that make up theblocking oligonucleotide adaptors can include one or more primersequences for amplification or sequencing.

In one example, a pair of blocking oligonucleotide adaptors (left andright) each comprises a first, second and third single-strandedoligonucleotide having an overhang portion that is 12 nucleotides inlength. The annealed adaptor-joining ends of left and right adaptors canhave two nicks, with one nick on opposite strands (e.g., see FIGS. 1Aand B).

In some embodiments, the left and right adaptors can comprise thefollowing sequences:

Left adaptor 1:

1^(st) strand: (24-mer)  (SEQ ID NO: 1) 5′-CTGCTGTACCGTACATCCGCCTTG-3′2^(nd) strand: (12-mer)  (SEQ ID NO: 2) 3′-GACGACATGGCA-5′3^(rd) strand: (11-mer)  (SEQ ID NO: 3) 3′-TGTAGGCGGAA-5′

Right adaptor 1:

3^(rd) strand: (11-mer)  (SEQ ID NO: 4) 5′-CATCCGCCTTG-3′2^(nd) strand: (12-mer)  (SEQ ID NO: 5) 5′-GCCGTACAGCAG-3′1^(st) strand: (24-mer)  (SEQ ID NO: 6) 3′-TGTAGGCGGAACCGGCATGTCGTC-5′

Annealed right and left adaptors (strands 1 and 2):

(SEQ ID NO: 7)                            ▾(nick)5′-CTGCTGTACCGTACATCCGCCTTG GCCGTACAGCAG-3′ (SEQ ID NO: 8)3′-GACGACATGGCA TGTAGGCGGAACCGGCATGTCGTC-5′                ▴(nick)

In some embodiments, a pair of a left adaptor 1 and a right adaptor 1can be annealed together to form a first strand having a sequence:

(SEQ ID NO: 9) 5′-CTGCTGTACCGTACATCCGCCTTGGCCGTACAGCAG-3′

In some embodiments, a pair of a left adaptor 1 and a right adaptor 1can be annealed together to form a second strand having a sequence:

(SEQ ID NO: 10) 3′-GACGACATGGCATGTAGGCGGAACCGGCATGTCGTC-5′

Left adaptor 2:

3^(rd) strand: (12-mer)  (SEQ ID NO: 11) 5′-ACATCCGCCTTG-3′2^(nd) strand: (9-mer)  (SEQ ID NO: 12) 5′-GTACAGCAG-3′1^(st) strand: (24-mer)  (SEQ ID NO: 13)3′-GCATGTAGGCGGAACCGGCATGTCGTC-5′

Right adaptor 2:

1^(st) strand: (27-mer)  (SEQ ID NO: 14)5′-CTGCTGTACCGTACATCCGCCTTGGCC-3′ 2^(nd) strand: (9-mer) (SEQ ID NO: 15) 3′-GACGACATG-5′ 3^(rd) strand: (12-mer)  (SEQ ID NO: 16)3′-TGTAGGCGGAAC-5′

In some embodiments, a pair of a left adaptor 2 and a right adaptor 2can be annealed together to form a first strand having a sequence:

(SEQ ID NO: 17) 5′-CTGCTGTACCGTACATCCGCCTTGGCCGTACAGCAG-3 ′

In some embodiments, a pair of a left adaptor 2 and a right adaptor 2can be annealed together to form a second strand having a sequence:

(SEQ ID NO: 18) 3′-GACGACATGGCATGTAGGCGGAACCGGCATGTCGTC-5′

In some embodiments, adaptors can include a 3′ overhang portion.

Left adaptor 3:

(SEQ ID NO: 1) 5′-Phos-CTGCTGTACCGTACATCCGCCTTG-3′ (SEQ ID NO: 2)3′-GACGACATGGCA-5′

Right adaptor 3:

(SEQ ID NO: 19) 5′-Phos-CTGCTGTACGGCCAAGGCGGATGT-3′ (SEQ ID NO: 20)3′-GACGACATGCCG-5′

In some embodiments, adaptors can include a palindromic ornon-palindromic sequence.

(SEQ ID NO: 21) 5′-Phos-ATTATAATTGCGGCCGC-3′ (SEQ ID NO: 22)3′-TAATATTAA-5′

In some embodiments, a nick can be a site located on one strand of adouble-stranded nucleic acid where the site lacks a phosphodiester bondbetween adjacent nucleotides, while the other strand has adjacentnucleotides joined by a phosphodiester bond at that same location. Insome embodiments, a phosphodiester bond can be replaced with analoglinkages that join adjacent nucleotides (or nucleotide analogs). In someembodiments, a gap can be a region on one strand of a double strandedpolynucleotide that is missing one or more nucleotide resides. In someembodiments, the nucleotide residue at the 3′ end of the gap can lack a5′ phosphate residue. In some embodiments, in step (b), one or bothstrands of the polynucleotide of interest is/are joined to the blockingoligonucleotide adaptor. For example, when one strand of adouble-stranded polynucleotide is joined to a blocking oligonucleotideadaptor, a nicked can be formed.

The terms “binding partner(s)” and “binding partner moiet(ies)” can beused interchangeably and in some embodiments, refers to two molecules,or portions thereof, which have a specific binding affinity for oneanother and typically will bind to each other in preference to bindingto other molecules. Typically, binding partners can be polypeptides thatcan bind or associate with each other. Interactions between the bindingpartners can be strong enough to allow enrichment and/or purification ofa conjugate that comprises a binding partner and a molecule associatedwith it (e.g., a biotinylated blocking oligonucleotide adaptor). Anexample of commonly used binding partners includes biotin andstreptavidin. Other examples include: biotin or desthiobiotin orphotoactivatable biotin and their binding partners avidin, streptavidin,Neutravidin™, or Captavidin™. Another binding partner for biotin can bea biotin-binding protein from chicken (Hytonen, et al., BMC StructuralBiology 7:8). Other examples of molecules that function as bindingpartners include: His-tags which bind with nickel, cobalt or copper;Ni-NTA which binds cysteine, histidine, or histidine patch; maltosewhich binds with maltose binding protein (MBP); lectin-carbohydratebinding partners; calcium-calcium binding protein (CBP); acetylcholineand receptor-acetylcholine; protein A and anti-FLAG antibody; GST andglutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNAglycosylase inhibitor) protein; antigen or epitope tags which bind toantibody or antibody fragments, particularly antigens such asdigoxigenin, fluorescein, dinitrophenol or bromodeoxyuridine and theirrespective antibodies; mouse immunoglobulin and goat anti-mouseimmunoglobulin; IgG bound and protein A; receptor-receptor agonist orreceptor antagonist; enzyme-enzyme cofactors; enzyme-enzyme inhibitors;and thyroxine-cortisol.

Methods for Adaptor-Polynucleotide Joining:

Provided herein are methods for joining together the ends of one or morepolynucleotides of interest to one or more blocking oligonucleotideadaptors. For example, methods include joining together one end of apolynucleotide of interest to a blocking oligonucleotide adaptor, orjoining together each end of a polynucleotide of interest to a separateblocking oligonucleotide adaptor. Joining together blockingoligonucleotide adaptors and polynucleotides of interest can be used aspart of a nucleic acid workflow for preparing one or more nucleic acidconstructs.

FIGS. 1A-B and 2 show some embodiments of blocking oligonucleotideadaptors in which left and right blocking oligonucleotide adaptors arejoined to a single polynucleotide of interest. For the sake of clarity,the diagram shows only a portion of the polynucleotide(s) of interest.

In some embodiments, methods for joining blocking oligonucleotideadaptors to polynucleotides of interest comprise: joining a linearpolynucleotide of interest at its first end to a first oligonucleotideadaptor, and joining the linear polynucleotide of interest at its secondend to a second oligonucleotide adaptor, and circularizing the resultinglinear construct. A “first end” and a “second end” of a polynucleotidecan refer to the 5′ end or the 3′ end of a polynucleotide of interest.Either the first end or second end of a polynucleotide can be the 5′ endor the 3′ end of the polynucleotide. In some embodiments, thecircularized construct can include nicks on opposite nucleic acidstrands.

In some embodiments, methods for preparing mate pair constructs and matepair libraries comprise joining each end of a linear polynucleotide ofinterest to a blocking oligonucleotide adaptor which comprise adouble-stranded oligonucleotide adaptor (duplex) having an overhangcohesive portion that anneals with a blocking oligonucleotide which canbe a separate single-stranded oligonucleotide. In some embodiments, theblocking oligonucleotide adaptor comprises (i) a first and a secondsingle-stranded oligonucleotide annealed together to form a duplexhaving an overhang portion and (ii) a third single-strandedoligonucleotide annealed to the overhang portion, wherein the overhangportion of the first and second double-stranded oligonucleotide adaptorare capable of annealing with each other, and wherein for the first andsecond double-stranded oligonucleotide adaptors the end of the duplexhaving the overhang portion forms an adaptor-joining end and the otherend of the duplex forms a target-joining end. In some embodiments,circularizing the linear construct includes removing the thirdsingle-stranded oligonucleotide (or a single-stranded portion of anoligonucleotide) from the overhang portions of the first and/or secondoligonucleotide adaptors so as to expose the overhang portions of theadaptor-joining ends. In some embodiments, the exposed overhang ends oftwo separate blocking oligonucleotide adaptors can be annealed together,thereby joining together the adaptor-joining ends of the first andsecond blocking oligonucleotide adaptors so as to circularize the linearconstruct. In some embodiments, the adaptors can hybridize withoutcomplete overlap between the overhang ends, so as to leave a gap or nickon one or both strands at the junction between the adaptor-joining endsof the first and second adaptors (FIGS. 1A and B-3). In someembodiments, the circularized construct can include nicks on oppositestrands. In some embodiments, removing the third single-strandedoligonucleotide from the overhang portions can include a denaturationstep using any combination of: elevated temperature, decrease/increasesalt concentration (e.g., sodium), and/or formamide.

In some embodiments, methods for preparing a mate pair constructs andmate pair libraries comprise: (a) providing one or more blockingoligonucleotide adaptors; (b) joining one or both ends of apolynucleotide of interest fragment to a separate blockingoligonucleotide adaptor to generate an adaptor-fragment construct; and(c) circularizing the adaptor-fragment construct to generate acircular-adaptor construct having at least one nick in the adaptorregion on at least one strand. In some embodiments, the circularizedconstruct can include at least two nicks, one each on opposite strands.In some embodiments, the method further comprises: (d) moving theposition of the at least one nick to a new position within thepolynucleotide of interest. In some embodiments, the method furthercomprises: (e) releasing a mate pair construct from the nicked constructby cleaving the strand opposite the at least one nick at the newposition.

In some embodiments, methods for preparing mate pair constructs and matepair libraries comprise: (a) providing a first and a seconddouble-stranded oligonucleotide adaptor each having (i) a first and asecond single-stranded oligonucleotide annealed together to form aduplex having an overhang portion and (ii) a third single-strandedoligonucleotide annealed to the overhang portion, wherein the overhangportion of the first and second double-stranded oligonucleotide adaptorsare capable of annealing with each other, and wherein for the first anda second double-stranded oligonucleotide adaptors the end of the duplexhaving the overhang portion forms an adaptor-joining end and the otherend of the duplex forms a target-joining end; (b) joining thetarget-joining end of the first double-stranded oligonucleotide adaptorto at least one strand of the first end of the linear double-strandedpolynucleotide of interest; (c) joining the target-joining end of thesecond double-stranded oligonucleotide adaptor to at least one strand ofthe second end of the linear double-stranded polynucleotide of interest;(d) removing the third single-stranded oligonucleotide from the firstand the second double-stranded oligonucleotide adaptors so as to exposethe overhang portions of the adaptor-joining ends; and (e) annealing theexposed overhang portions of the adaptor-joining ends thereby joiningtogether the adaptor-joining ends of the first and second blockingoligonucleotide adaptors so as to circularize the linear construct. Insome embodiments, the circular polynucleotide of interest comprises atleast one nick at a junction between the adaptor-joining ends of thefirst and the second double-stranded oligonucleotide adaptors that wereannealed together in step (e). In some embodiments, the circularizedconstruct can include at least two nicks, one each on opposite strands.In some embodiments, annealing the adaptor-joining ends of a first andsecond blocking oligonucleotide adaptor can include a hybridization stepusing any combination of: elevated temperature, decrease/increase saltconcentration (e.g., sodium), and/or formamide. In some embodiments,removing the third single-stranded oligonucleotide from the overhangportions can include a denaturation step using any combination of:elevated temperature, increase/decrease salt concentration (e.g.,sodium), and/or formamide.

In some embodiments, a barcode sequence can be joined to apolynucleotides of interest and/or to a blocking oligonucleotide adaptorto generate barcoded mate pair constructs and barcoded mate pairlibraries (FIG. 7). For example, methods for preparing barcoded matepair constructs and barcoded mate pair libraries can comprise: (a)providing one or more types of barcode adaptors (e.g., BC1 and BC2) andone or more types of blocking oligonucleotide adaptors; (b) joining oneor both ends of a polynucleotide of interest to a barcode adaptor togenerate a barcode-fragment construct; (c) joining one or both ends ofthe barcode-fragment construct to a blocking oligonucleotide adaptor togenerate a blocking oligonucleotide-barcode-fragment construct; and (d)circularizing the blocking oligonucleotide-barcode-fragment construct.

In some embodiments, preparing a barcoded mate pair construct comprises:(a) providing a first and second double-stranded barcoded adaptor; (b)providing a first and a second double-stranded oligonucleotide adaptoreach having (i) a first and a second single-stranded oligonucleotideannealed together to form a duplex having an overhang portion and (ii) athird single-stranded oligonucleotide annealed to the overhang portion,wherein the overhang portion of the first and second double-strandedoligonucleotide adaptors are capable of annealing with each other, andwherein for the first and a second double-stranded oligonucleotideadaptors the end of the duplex having the overhang portion forms anadaptor-joining end and the other end of the duplex forms atarget-joining end; (c) joining the first double-stranded barcodedadaptor to the first end of a linear double-stranded polynucleotide ofinterest; (d) joining the second double-stranded barcoded adaptor to thesecond end of the linear double-stranded polynucleotide of interest; (e)joining the target-joining end of the first double-strandedoligonucleotide adaptor to the first barcoded adaptor; (f) joining thetarget-joining end of the second double-stranded oligonucleotide adaptorto the second barcoded adaptor; (g) removing the third single-strandedoligonucleotide from the first and the second double-strandedoligonucleotide adaptors so as to expose the overhang portions of theadaptor-joining ends; and (h) annealing the exposed overhang portions ofthe adaptor-joining ends thereby joining together the adaptor-joiningends of the first and second blocking oligonucleotide adaptors so as tocircularize the barcoded linear polynucleotide of interest. In someembodiments, the overhang portions of first and second double-strandedoligonucleotide adaptors are capable of annealing with each other. Insome embodiments, the barcoded circular polynucleotide of interestcomprises at least one nick at a junction between the adaptor-joiningends of the first and the second double-stranded oligonucleotideadaptors annealed together in step (h).

Releasing a Mate Pair Construct

Moving a Nick:

In some embodiments, methods for preparing mate pair constructs andbarcoded mate pair constructs further comprises the step: moving the atleast one nick to a different/new position within a circularizedconstruct. In some embodiments, the at least one nick can be moved to anew position within the double-stranded polynucleotide of interest. Forexample, the at least one nick can be moved to a new position byconducting a nick translation reaction, or by conducting a combinationof exonuclease and strand extension reactions, or by conducting acombination of an exonuclease and cleavage reactions. A nick translationreaction or exonuclease reaction can proceed to the end of thepolynucleotide of interest, or can stop at any position along thepolynucleotide of interest.

Moving a Nick with a Nick Translation Reaction:

In some embodiments, a nick can be moved to a new position within thedouble-stranded polynucleotide of interest by conducting a nicktranslation reaction. For example, a nick translation reaction can be acoupled 5′ to 3′ DNA polymerization/degradation reaction, or a coupled5′ to 3′ DNA polymerization/strand displacement reaction. A nicktranslation reaction can proceed in a 5′ to 3′ direction on the nucleicacid strand having a nick.

In some embodiments, moving the at least first nick comprises performinga nick translation reaction on the circularized polynucleotide ofinterest. In some embodiments, the nick translation reaction can beperformed using deoxyribonucleoside triphosphates and an enzyme selectedfrom the group consisting of E. coli DNA polymerase I, Taq DNApolymerase, Vent DNA polymerase, Klenow DNA polymerase I, Tfi DNApolymerase, Bst DNA polymerase, and phi29 DNA polymerase.

In some embodiments, the at least one nick can be moved to a newposition that is less than about 500 bases, or about 400 bases, or about300 bases, or about 200 bases, or about 100 bases within thepolynucleotide of interest. In some embodiments, the at least one nickcan be moved about 25-50 bases, or about 50-75 bases, or about 75-100bases, or about 100-125 bases, or about 125-150 bases, or about 150-175bases, or about 175-200 bases, or about 200-300 bases, or about 300-400bases, or about 400-500 bases, or more. Thus, the length of thepolynucleotide of interest in the released mate pair construct can bemodulated by adjusting the length of time that the nick translationreaction is permitted to occur.

In some embodiments, methods for preparing mate pair constructs andbarcoded mate pair constructs further comprise the step: opening the atleast one nick into a gap. In some embodiments, the at least one nickcan be opened into a gap by conducting an exonuclease reaction so as toremove at least a portion of the strand having the nick. In someembodiments, a nick can be opened to a gap with an exonuclease which canbe a T7 exonuclease, lambda exonuclease, E. coli exonuclease III, DNase,or an ATP-dependent DNase.

In some embodiments, methods for preparing mate pair constructs andbarcoded mate pair constructs further comprise the step: cleaving thestrand opposite the gap so as to release a linear mate pair construct ora barcoded mate pair construct. In some embodiments, the strand oppositethe gap can be cleaved with a single-strand specific endonuclease enzymeto release a linear mate pair construct or a barcoded mate pairconstruct. In some embodiments, the circular polynucleotide of interestcan be cleaved with a single-strand specific endonuclease enzymeopposite the new position of the nick (or gap) to release a linear matepair. In some embodiments, the cleaving can be performed by an enzymeselected from the group consisting of S1 nuclease, mung bean nuclease,nuclease P1, nuclease BAL-31 and nucleases isolated from Neurosporacrassa or Ustilago maydis.

Moving a Nick with Exonuclease and Strand Extension Reactions:

In some embodiments, the moving the at least first nick to a newposition can comprise conducting an exonuclease reaction at the nick,and a nucleic acid strand extension reaction.

For example, moving the at least first nick can comprise: (a) conductingan exonuclease reaction on the at least first nick into thepolynucleotide of interest so that at least a portion of thepolynucleotide of interest is single-stranded (and leaving a terminal 3′end at or near the original location of the at least first nick); and(b) conducting a nucleic acid strand extension reaction at the terminal3′ end and stopping the strand extension reaction prior to the stopposition of the exonuclease reaction, so as to leave a portion of thepolynucleotide of interest single-stranded. A linear mate pair constructcan be released by cleaving the single-stranded portion of thepolynucleotide of interest with a single-stranded specific endonucleaseenzyme. In some embodiments, an exonuclease reaction can remove aportion or the entire region of the polynucleotide of interest.

In some embodiments, the exonuclease can be conducted with a 5′ to 3′exonuclease, such as T7 exonuclease or lambda exonuclease.

In some embodiments, the DNA strand extension reaction can be conductedusing a DNA polymerase enzyme.

In some embodiments, a linear mate pair construct can be released bycleaving the single-stranded portion of the polynucleotide of interestwith a single-strand specific endonuclease, such as S1 nuclease, mungbean nuclease, P1 nuclease and/or BAL 31 nuclease.

The length of time of the exonuclease and/or strand extensionreaction(s) can be modulated to increase or decrease the distance tomove the at least one nick to a new position within the polynucleotideof interest.

Moving a Nick with Exonuclease Reactions:

In some embodiments, the moving the at least first nick to a newposition can comprise: (a) conducting an exonuclease reaction on the atleast first nick so that at least a portion of the polynucleotide ofinterest is single-stranded; and (b) cleaving the single-strandedportion of the polynucleotide of interest with a single-strandedspecific endonuclease enzyme so as to release a linear mate pairconstruct.

In some embodiments, the exonuclease can be conducted with a 5′ to 3′exonuclease, such as T7 exonuclease or lambda exonuclease. In someembodiments, an exonuclease reaction can remove a portion or the entireregion of the polynucleotide of interest.

In some embodiments, the single-strand specific endonuclease can be anS1 nuclease, mung bean nuclease, P1 nuclease or BAL 31 nuclease.

Embodiments of Methods

Provided herein are various embodiments for practicing methods of thepresent teachings. In some embodiments, a workflow for preparing a matepair library (with or without barcoded adaptors) can include fragmentingthe polynucleotide of interest. In some embodiments, the fragmentedpolynucleotide of interest can be further manipulated, including anycombination and in any order: size selection, end repair, adaptorligation or oligonucleotide ligation (e.g., blocking oligonucleotideadaptors, barcodes adaptors, P1, P2 or A adaptors), adaptor annealing,circularization, moving-a-nick, releasing a linear mate pair construct,nick translation, tailing, amplification, purification, removing linearnucleic acids, washing, quantization, immobilization, and/ordenaturation. In some embodiments, additional nucleic acid manipulationsteps can include: exonuclease or endonuclease reactions. Any of thesesteps can be omitted or repeated. In some embodiments, nucleic acidmanipulation steps can be conducted under suitable conditions, andinclude ATP, nucleotides (e.g., dNTPs) or nucleosides, salts, magnesium,manganese, or sodium, or be conducted under suitable pH or temperatures.

In some embodiments, one end of a first polynucleotide of interest canbe joined to a first blocking oligonucleotide adaptor, and one end of asecond polynucleotide of interest can be joined to a second blockingoligonucleotide adaptor, where the first and second blockingoligonucleotide adaptors can be the same or different.

In some embodiments, polynucleotides of interest can be joined toblocking oligonucleotide adaptors and/or to barcoded adaptors with aligase enzyme (e.g., T4 DNA ligase).

In some embodiments, methods for preparing a mate pair library cancomprise: (A) fragmenting a polynucleotide of interest to generatepolynucleotide fragments; (B) size-selecting the polynucleotidefragments; (C) repairing the ends of the polynucleotide fragments togenerate repaired polynucleotide fragments; (D) joining each end of therepaired polynucleotide fragments to a biotinylated blockingoligonucleotide adaptor to generate adaptor-fragment constructs, whereinthe biotinylated blocking oligonucleotide adaptors include (i) a firstand a second single-stranded oligonucleotide annealed together to form aduplex having an overhang portion and (ii) a third single-strandedoligonucleotide annealed to the overhang portion, wherein the overhangportions of the first and second double-stranded oligonucleotide adaptorare capable of annealing with each other, and wherein for the first andsecond double-stranded oligonucleotide adaptors the end of the duplexhaving the overhang portion forms an adaptor-joining end and the otherend of the duplex forms a target-joining end, and wherein the ends ofthe repaired polynucleotide fragments are joined to the target-joiningend of the blocking oligonucleotide adaptors, and wherein the first orsecond single-stranded oligonucleotides include at least one biotinmoiety; (E) removing the third single-stranded oligonucleotides from theoverhang portions so as to expose the overhang portions; (F) annealingtogether the overhang portions of the biotinylated blockingoligonucleotide adaptors so as to join the adaptor-joining ends therebycircularizing the polynucleotide fragments, wherein the junctionsbetween the adaptor-joining ends includes a nick, and wherein the nicksare located on opposite nucleic acid strands; (G) conducting a nicktranslation reaction on the nicks so as to move the nicks to a newposition within the polynucleotide fragments; (H) stopping the nicktranslation reaction; (I) conducting an exonuclease reaction at thenicks at the new position so as to open the nicks into gaps; (J)cleaving the strands opposite the gaps with a single-strand specificendonuclease so as to release linear mate pair constructs; (K) adding anon-template nucleotide tail (e.g., A-tail) to the 3′ ends of the linearmate pair constructs to generate tailed mate pair constructs; and (L)joining each end of the tailed mate pair constructs to tailed adaptors(e.g., T-tail) having amplification primer sequences and/or sequencingprimer sequences (e.g., P1, P2 and/or A adaptors) so as to generateprimer-mate-pair constructs having nicks at the junctions between thetailed adaptors and the tailed mate pair constructs; (M) conducting anick translation to close the nick(s); and (N) amplifying the mate pairconstruct of step (M).

In some embodiments, an end-repair reaction (step C) can be performedbefore size-selection (step B). Conducting an end-repair reaction priorto size-selection can improve isolation of larger polynucleotidefragments for preparation of a mate pair library. In some embodiments,conducting an end-repair reaction prior to size-selection can yield anarrower size range for larger polynucleotide fragments such as about4-6 kb, or about 6-8 kb, or about 8-10 kb, or about 10-12 kb, or about12-14 kb, or about 14-16 kb, or about 16-18 kb, or about 18-20 kb.

In some embodiments, released mate pair constructs (e.g. at step (J))can be subjected to an end-repair reaction to generate blunt or overhangends. In some embodiments, the tailing step (K) can be omitted. In someembodiments, amplification primer adaptors and/or sequencing primeradaptors (e.g., at step L)) need not be tailed adaptors. In someembodiments, joining amplification primer adaptors and/or sequencingprimer adaptors (e.g., at step (L)) need not generate a nick at thejunction between the adaptors and mate pair constructs. Accordingly, anick translation step (e.g., step (M)) need not be performed.

Joining Together Two Polynucleotides of Interest

Provided herein are methods for joining together two polynucleotides ofinterest using blocking oligonucleotide adaptors. For example, one endof a first polynucleotide of interest can be joined to a first blockingoligonucleotide of interest and one end of a second polynucleotide ofinterest can be joined to a second blocking oligonucleotide of interest,and the overhang portions of the first and second blockingoligonucleotide adaptors can be exposed to permit annealing between thetwo overhang portions thereby joining together the first and secondpolynucleotides of interest.

In some embodiments, a first polynucleotide of interest can be joined atits first end to a first blocking oligonucleotide adaptor and a secondpolynucleotide of interest can be joined at its first end to a secondblocking oligonucleotide adaptor. In some embodiments, the first andsecond blocking oligonucleotide adaptors have a third single-strandedoligonucleotides (or a single-stranded portion of an oligonucleotide)that anneals to the overhang portions. In some embodiments, the thirdsingle-stranded oligonucleotides (of the first and second blockingoligonucleotide adaptors) can be removed to expose the overhangportions. In some embodiments, the overhang portions can anneal togetherthereby joining together the first and second polynucleotides ofinterest.

In some embodiments, the first and second blocking oligonucleotideadaptors can be joined to first and second polynucleotides of interest,respectively, by hybridization and/or enzymatic ligation.

In some embodiments, one or more junctions between the annealed overhangportions can include a nick or gap. In some embodiments, the nick can beligated to covalently join together the first and second blockingoligonucleotide adaptors, thereby joining together the first and secondpolynucleotides of interest. In some embodiments, a nick translationreaction can be conducted on the nick(s). In some embodiments, the nicktranslation reaction can move in the direction towards and within thepolynucleotide of interest. In some embodiments, the nick translationreaction can move to the end(s) of the polynucleotide of interest so asto covalently join together the first and second polynucleotides ofinterest.

Polynucleotides of Interest

The terms “polynucleotide(s) of interest” and “polynucleotidefragment(s)” can be used interchangeably and refer to nucleic acids thatare being analyzed, characterized or manipulated. In some embodiments,polynucleotides of interest can include single-stranded anddouble-stranded nucleic acids. In some embodiments, polynucleotides ofinterest can include DNA, RNA or chimeric RNA/DNA. In some embodiments,polynucleotides of interest can be isolated in any form includingchromosomal, genomic, organellar (e.g., mitochondrial, chloroplast orribosomal), recombinant molecules, cloned, subcloned, amplified (e.g.,PCR amplified), cDNA, RNA such as precursor mRNA or mRNA,oligonucleotide, or any type of nucleic acid library. In someembodiments, polynucleotides of interest can be isolated from any sourceincluding from organisms such as prokaryotes, eukaryotes (e.g., humans,plants and animals), fungus, and viruses; cells; tissues; normal ordiseased cells or tissues or organs, body fluids including blood, urine,serum, lymph, tumor, saliva, anal and vaginal secretions, amnioticsamples, perspiration, and semen; environmental samples; culturesamples; or synthesized nucleic acid molecules prepared usingrecombinant molecular biology or chemical synthesis methods. In someembodiments, polynucleotides of interest can be chemically synthesizedto include any type of natural and/or analog nucleic acid. In someembodiments, polynucleotides of interest can be isolated from aformalin-fixed tissue, or from a paraffin-embedded tissue, or from aformalin-fix paraffin-embedded (FFPE) tissue. In some embodiments,polynucleotides of interest can be associated with counter ions,including H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺ or Na⁺. In some embodiments,the amount of starting material (e.g., polynucleotide of interest) canbe about 1 ng-50 ug, or about 1 ng-1 ug, or about 1 ug-5 ug, or about 5ug-25 ug, or about 25 ug-50 ug.

Fragmentation Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a fragmenting step. One or morepolynucleotides of interest can be fragmented using mechanical stress,including without limitation: shearing forces, fluid shear, hydrodynamicshear, or pulsatile shear. Mechanical stress can be applied to apolynucleotide of interest by: sonication, nebulization, or cavitation.Mechanical stress can randomly fragment nucleic acids. A polynucleotideof interest can be fragmented with enzymatic reactions, such as: anytype I, type II, type IIs, type IIB, type III or type IV restrictionendonucleases; any nicking endonuclease restriction enzymes;endonuclease (e.g., DNase I); and/or exonuclease enzymes. Apolynucleotide of interest can be fragmented with an enzyme cocktail,for example Fragmentase™ (New England Biolabs). A polynucleotide ofinterest can be fragmented with a transposase and transposable element,for example Nextera™ technology from Epicentre. A polynucleotide ofinterest can be fragmented using any chemical reactions, including:dimethyl sulfate; hydrazine, NaCl, piperidine, or acid. A polynucleotideof interest can be fragmented using any type of high energy radiation,such as ultraviolet radiation. In some embodiments, polynucleotides ofinterest can be fragmented to a size range of about 200 bp-1 kb, orabout 500 bp-1 kb, or about 700 bp-1 kb, or about 1 kb-2 kb, or about 1kb-3 kb, or about 1 kb-4 kb, or about 1 kb-5 kb, or about 1 kb-6 kb, orabout 1 kb-7 kb or larger.

Size Selection Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a size-selecting step to obtainpolynucleotide fragments having any desired size or size range. In someembodiments, polynucleotide fragments are not size-selected. A nucleicacid size selection method can include without limitation: solid phaseadherence or immobilization; electrophoresis, such as gelelectrophoresis; and chromatography, such as HPLC and size exclusionchromatography. A solid phase adherence/immobilization method cantypically involve micro paramagnetic beads coated with a chemicalfunctional group that interacts with nucleic acids under certain ionicstrength conditions with or without polyethylene glycol or polyalkyleneglycol.

Examples of solid phase adherence/immobilization methods include but arenot limited to; SPRI (Solid Phase Reversible Immobilization) beads fromAgencourt (see Hawkins 1995 Nucleic Acids Research 23:22) which arecarboxylate-modified paramagnetic beads; MagNA Pure™ magnetic glassparticles (Roche Diagnostics, Hoffmann-La Roche Ltd.); MagneSil™magnetic bead kit from Promega; Bilatest™ magnetic bead kit from BilatecAG; Magtration™ paramagnetic system from Precision System Science, Inc.;Mag-Bind™ from Omega Bio-Tek; MagPrep™ silica from Merck/Estapor; SNARe™DNA purification system from Bangs; and Chemagen™ M-PVA beads fromChemagen.

In some embodiments, size-selected polynucleotide fragments can be about50-3000 base pairs in length, or about 50-2000 base pairs in length, orabout 50-1500 base pairs in length, or about 50-1000 base pairs inlength, or about 50-700 base pairs in length. In some embodiments,size-selected nucleic acid fragments can be about 50-150 bases, or about150-250 bases, or about 250-500 bases, or about 500-1000 bases, or about1000-2000 bases in length. In some embodiments, size-selected nucleicacid fragments can be about 1-3 kb, or about 3-6 kb, or about 6-10 kb,or about 10-15 kb, or about 15-20 kb, or about 20-25 kb, or about 25-30kb, or about 30-40 kb, or about 40-50 kb, or about 50-75 kb, or about75-100 kb, or about 100-150 kb, or about 150-200 kb, or longer.

Repairing Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a repair step (e.g., “end repairing”or “repairing the ends” or “repair”). Polynucleotide fragments can befragmented to generate nucleic acid fragments having a first end and asecond end. A fragmenting step can generate first ends, second ends, orinternal portions, having undesirable features, such as nicks, overhangends, ends lacking a phosphorylated end, ends having a phosphorylatedend, or nucleic acid fragments having apurinic or apyrimidinic residues.Polynucleotide fragments can be repaired at one or both ends, and/orrepaired at an internal region. In some embodiments, enzymatic reactionscan be conducted to repair one or more ends or internal portions. Insome embodiments, enzymatic reactions can be conducted to convertoverhang ends to blunt ends, or to phosphorylate or de-phosphorylate the5′ end of a strand, or to close nicks, or to repair oxidized guanines orpyrimidines, or to repair deaminated cytosines, or to hydrolyze theapurinic or apyrimidinic residues. In some embodiments, an enzymaticreaction can generate an overhang end (e.g., sticky end). For example,restriction endonucleases or a tailing enzyme can generate an overhangend.

In some embodiments, repairing or end-repairing nucleic acid fragmentsincludes contacting nucleic acid fragments, or contacting a plurality offirst ends and/or second ends with: an enzyme to close single-strandednicks in duplex DNA (e.g., T4 DNA ligase); an enzyme to phosphorylatethe 5′ end of at least one strand of a duplex DNA (e.g., T4polynucleotide kinase); an enzyme to remove a 5′ phosphate (e.g., anyphosphatase enzyme, such as calf intestinal alkaline phosphatase,bacterial alkaline phosphatase, shrimp alkaline phosphatase, Antarcticphosphatase, and placental alkaline phosphatase); an enzyme to remove 3′overhang ends (e.g., DNA polymerase I, Large (Klenow) fragment, T4 DNApolymerase, mung bean nuclease); an enzyme to fill-in 5′ overhang ends(e.g., T4 DNA polymerase, Tfi DNA polymerase, Tli DNA polymerase, TaqDNA polymerase, Large (Klenow) fragment, phi29 DNA polymerase, Mako DNApolymerase (Enzymatics, Beverly, Mass.), or any heat-stable orheat-labile DNA polymerase); an enzyme to remove 5′ overhand ends (e.g.,S1 nuclease); an enzyme to remove 5′ or 3′ overhang ends (e.g., mungbean nuclease); an enzyme to hydrolyze single-stranded DNA (e.g.,nuclease P1); an enzyme to remove both strands of double-stranded DNA(e.g., nuclease Bal-31); and/or an enzyme to remove an apurinic orapyrimidinic residue (e.g., endonuclease IV). In some embodiments, apolymerase can have exonuclease activity, or have a reduced or lack ofnuclease activity.

A repairing or end-repairing reaction can be supplemented withadditional repairing enzymes in any combination and in any amount,including: endonuclease IV (apurinic-apyrimidinic removal), Bst DNApolymerase (5′>3′ exonuclease for nick translation) formamidopyrimidineDNA glycosylase (FPG) (e.g., base excision repair for oxidized purines)uracil DNA glycosylase (uracil removal), T4 endonuclease V (pyrimidineremoval) and/or endonuclease VIII (removes oxidized pyrimidines). Insome embodiments, a repairing or end-repairing reaction can be conductedin the presence of appropriate co-factors, including dNTPs, NAD,(NH₄)SO₄, KCl, and/or MgSO₄. In some embodiments, the additionalrepairing enzymes can be included in a repair or end-repairing reactionat any concentration, including: about 0.1-1 U/uL, or about 1-2 U/uL, orabout 2-3 U/uL, or about 3-4 U/uL, or about 5 U/uL, or about 5-10 U/uL,or about 10-15 U/uL, or about 15-20 U/uL, or more.

In some embodiments, a repairing or end-repairing step can be performedin the presence of appropriate buffers and/or nucleotides (includingnucleotide analogs or biotinylated nucleotides), and at an appropriatepH and temperature(s). A repairing or end-repairing step can beconducted in the presence of a nucleic acid damage-mitigatingcomposition.

Tailing Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a tailing step. One or morenon-template nucleotides can be enzymatically added to a first endand/or a second end of a nucleic acid (e.g., polynucleotide fragment, ora first or second single-stranded oligonucleotide). In some embodiments,a DNA polymerase can be used to add one or more non-template nucleotidesto a terminal 3′ end of a nucleic acid strand. In some embodiments, anon-proofreading DNA polymerase can be used to add a single non-templateA-residue to a 3′ end a of a nucleic acid strand. In some embodiments, aDNA polymerase can be a Taq DNA polymerase (or a derivative thereof). Insome embodiments, DNA polymerases having proofreading activity can beused to add a single non-template 3′ A-tail. In some embodiments, a DNApolymerase can be a Tfi (exo minus) DNA polymerase, large (Klenow)fragment (3′>5′ exo minus), or derivative polymerases thereof. In someembodiments, T4 DNA polymerase (e.g., exo-) can be used to add anon-template, single nucleotide residue to a 3′ end of a nucleic acidstrand. In some embodiments, a first end and/or a second end of anucleic acid lack a nucleotide tail.

Adaptor-Joining Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise an adaptor-joining step. Apolynucleotide fragment (e.g., fragments of polynucleotides of interest)can be joined to one or more nucleic acid adaptors by hybridization orenzymatic ligation to generate adaptor-fragment constructs.

In some embodiments, one end or both ends of nucleic acid fragments canbe joined to at least one type of adaptor. One or both ends of apolynucleotide fragment can be joined to at least one nucleic acidadaptor, including blocking oligonucleotide adaptors, barcoded adaptors,sequencing primer adaptors, amplification primer adaptors, universaladaptors and/or others.

In some embodiments, adaptors can be joined to one or both ends ofpolynucleotide fragments essentially simultaneously or sequentially. Forexample, each end of a polynucleotide fragment can be joined to adaptorsin a joining reaction (e.g., essentially simultaneous adaptor-joining).In another example, in a first step, one end of a polynucleotidefragment can be joined to a first adaptor, and in a second step theother end of the polynucleotide fragment can be joined to a secondadaptor (e.g., sequential adaptor-joining steps). A skilled artisan willappreciate that other combinations of adaptor-joining reactions can bepracticed.

In some embodiments, an adaptor can be single-stranded ordouble-stranded nucleic acids, or can include single-stranded ordouble-stranded portions. In some embodiments, an adaptor can have anystructure, including linear, hairpin, forked, or stem-loop.

In some embodiments, an adaptor can be a blocking oligonucleotideadaptor which comprises a double-stranded oligonucleotide adaptor(duplex) having an overhang cohesive portion of anneals with a blockingoligonucleotide which can be a separate single-stranded oligonucleotide.

In some embodiments, an adaptor can include nucleotide sequences thatare complementary to sequencing primers (e.g., P1, P2 and/or A),amplification primers, universal sequences and/or barcode sequences. Forexample, released mate pair constructs can be joined at each end to adifferent sequencing adaptor to prepare a nucleic acid library forsequencing with SOLiD™ sequencing reactions (WO 2006/084131) orsequencing with ion-sensitive sequencing reactions (e.g., Ion TorrentPGM™ sequencer from Life Technologies Corporation).

In some embodiments, an adaptor can have any length, including fewerthan 10 bases in length, or about 10-20 bases in length, or about 20-50bases in length, or about 50-100 bases in length, or longer.

In some embodiments, an adaptor can have any combination of blunt end(s)and/or sticky end(s). In some embodiments, at least one end of anadaptor can be compatible with at least one end of a nucleic acidfragment. In some embodiments, a compatible end of an adaptor can bejoined to a compatible end of a nucleic acid fragment. In someembodiments, an adaptor can have a 5′ or a 3′ overhang end. In someembodiments, an adaptor can have a 3′ overhang end with at least onephosphorothiolate, phosphorothioate, and/or phosphoramidate linkage. Insome embodiments, an adaptor can have a 3′ overhang end with at leasttwo phosphorothioate linkages.

In some embodiments, an adaptor can include a monomeric sequences (e.g.,AAA, TTT, CCC, or GGG) of any length, or an adaptor can include acomplex sequence (e.g., non-monomeric sequence), or can include bothmonomer and complex sequences.

In some embodiments, an adaptor can have a 5′ or 3′ tail. In someembodiments, the tail can be one, two, three, or more nucleotides inlength. In some embodiments, an adaptor can have a tail comprisingadenine, thymine, cytosine and/or guanine base (or analogs thereof). Insome embodiments, an adaptor can have a monomeric tail sequence of anylength. In some embodiments, at least one end of an adaptor can have atail that is compatible with a tail on one end of a nucleic acidfragment. In some embodiments, an adaptor can lack a terminal tail.

In some embodiments, an adaptor can include an internal nick. In someembodiments, an adaptor can have at least one strand that lacks aterminal 5′ phosphate residue. In some embodiments, an adaptor lacking aterminal 5′ phosphate residue can be joined to a nucleic acid fragmentto introduce a nick at the junction between the adaptor and the nucleicacid fragment.

In some embodiments, an adaptor can include one or more universal bases(e.g., inosine). In some embodiments, an adaptor can include one or moreribonucleoside residues. In some embodiments, an adaptor can be chimericRNA/DNA. In some embodiments, an adaptor can include at least onescissile linkage. In some embodiments, a scissile linkage can besusceptible to cleavage or degradation by an enzyme or chemicalcompound. In some embodiments, an adaptor can include one or more uracilresidues. In some embodiments, an adaptor can include at least onephosphorothiolate, phosphorothioate, and/or phosphoramidate linkage.

In some embodiments, an adaptor can include identification sequences. Insome embodiments, an identification sequence can be a unique sequence(e.g., barcode sequence). In some embodiments, an identificationsequence can be used for sorting or tracking. In some embodiments, amate pair construct can include one or more identification sequences(e.g., barcodes) that are the same or different. In some embodiments, abarcode sequence can allow identification of a particular adaptor amonga mixture of different adaptors having different barcodes sequences. Forexample, a mixture can include 2, 3, 4, 5, 6, 7-10, 10-50, 50-100,100-200, 200-500, 500-1000, or more different adaptors having uniquebarcode sequences. In some embodiments, a plurality of polynucleotidefragments which are joined to blocking oligonucleotide adaptors and atleast one unique identifier adaptor (e.g., barcoded adaptor) can bepooled together for any manipulation, including size selection, endrepair, adaptor ligation, adaptor annealing, circularization,moving-a-nick, releasing a linear mate pair construct, nick translation,tailing, amplification, purification, removing linear nucleic acids,washing, quantization, immobilization, denaturation and/or attachment toa solid surface. Examples of a polynucleotide fragment joined to abarcode are described in U.S. Ser. No. 13/026,046.

In some embodiments, adaptors can include any type of restriction enzymerecognition sequence, including type I, type II, type IIs, type IIB,type III or type IV restriction enzyme recognition sequences. Forexample, adaptors can include an Ecop151 or MmeI recognition site. Insome embodiments, adaptors can include a nicking restriction enzymesequence, including: EcoP15I, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI,Nt.AlwI, Nt.BbvCI, or Nt.BstNBI.

In some embodiments, the adaptors can include a cell regulationsequences, including a promoter (inducible or constitutive), enhancers,transcription or translation initiation sequence, transcription ortranslation termination sequence, secretion signals, Kozak sequence,cellular protein binding sequence, and the like.

In some embodiments, any component of a blocking oligonucleotide adaptorcan include a binding partner (e.g., biotin or streptavidin moiety) topermit separation from undesirable reagents in a reaction. In someembodiments, a first or a second single-stranded oligonucleotide, whichcan anneal to form a duplex structure having an overhang portion, caninclude a binding partner. In some embodiments, a third single-strandedoligonucleotide (a blocking oligonucleotide which anneals to an overhangend) can include a binding partner.

In some embodiments, one or more type(s) of adaptors can be joined topolynucleotide fragments, where the adaptors are present in a ligationreaction at about 10×-300× (or higher) relative to the amount of nucleicacid fragments. For example, adaptors can be present in an amount thatis about 10×, or about 20×, or about 30×, or about 50×, or about 75×, orabout 100×, or about 150×, or about 200×, or about 250×, or about 300×,or higher amounts compared to the amount of polynucleotide fragments. Insome embodiments, molar amounts of adaptors can be compared to molaramounts of polynucleotide fragments. One skilled in the art will readilyrecognize that other unit amounts of adaptors and polynucleotidefragments can be compared.

In some embodiments, a mate pair construct can include an adaptor havingamplification and/or sequencing primer sequences, such as those shown inTable 1 below.

TABLE 1 SEQ ID Adaptors: Sequence NOS: P1-Adaptor5′CCACTACGCCTCCGCTTTCCTCTCT 23 (top strand) ATGGGCAGTCGGTGFT3′P1-Adaptor 5′TCACCGACTGCCCATAGAGAGGAAA 24 (bottom strand)GCGGAGGCGTAGTGEOC3′ P2-LMP Adaptor 5′GAGAATGAGGAACCCGGGGCAEOC3′ 25(top strand) P2-LMP 5′CTGCCCCGGGTTCCTCATTCTOT3′ 26 Adaptor(bottom strand) PCR primer 1 5′CCACTACGCCTCCGCTTTCCTCTCT 27 ATG3′PCR primer 2 5′CTGCCCCGGGTTCCTCATTCT3′ 28 PCR primer 35′CCATCTCATCCCTGCGTGTC3′ 29 P1-adaptor ION 5′CCACTACGCCTCCGCTTTCCTCTCT30 (top strand) ATGGGCAGTCGGTGAT3′ P1-adaptor ION3′MMGGTGATGCGGAGGCGAAAGGAGA 31 (bottom strand) GATACCCGTCAGCCACTA5′A-adaptor 5′CCATCTCATCCCTGCGTGTCTCCGA 32 (top strand) CTCAG3′ A-adaptor3′MMGGTAGAGTAGGGACGCACAGAGG 33 (bottom strand) CTGAGTC5′ LEGEND: F =A-3′phosphorothioate E = G-3′phosphorothioate O = C-3′phosphorothioate M= T-3′ phosphorothioate

In some embodiments, a mate pair construct can include at least one ofthe identification sequences (e.g., barcodes) listed in Table 2 below.For example, a mate pair construct can be joined to an adaptorcomprising a barcode sequence. The identification sequences are shown ina 5′>3′ orientation.

TABLE 2 BC: Sequence: SEQ ID NO:  1 CCTCTTACAC SEQ ID NO. 34 2ACCACTCCCT SEQ ID NO. 35 3 TATAACCTAT SEQ ID NO. 36 4 GACCGCATCCSEQ ID NO. 37 5 CTTACACCAC SEQ ID NO. 38 6 TGTCCCTCGC SEQ ID NO. 39 7GGCATAACCC SEQ ID NO. 40 8 ATCCTCGCTC SEQ ID NO. 41 9 GTCGCAACCTSEQ ID NO. 42 10 AGCTTACCGC SEQ ID NO. 43 11 CGTGTCGCAC SEQ ID NO. 44 12TTTTCCTCTT SEQ ID NO. 45 13 GCCTTACCGC SEQ ID NO. 46 14 TCTGCCGCACSEQ ID NO. 47 15 CATTCAACTC SEQ ID NO. 48 16 AACGTCTCCC SEQ ID NO. 49 17GCGGTGAGCC SEQ ID NO. 50 18 TCATCCGCCT SEQ ID NO. 51 19 CAGTTACCATSEQ ID NO. 52 20 AAAGCTTGAC SEQ ID NO. 53 21 GGAACCGCAC SEQ ID NO. 54 22TCATCTTCTC SEQ ID NO. 55 23 CAAGCACCGC SEQ ID NO. 56 24 ATACCGACCCSEQ ID NO. 57 25 TCATCATGTT SEQ ID NO. 58 26 CGGGCTCCCG SEQ ID NO. 59 27AAGTTTGCTG SEQ ID NO. 60 28 GTAGTAAGCT SEQ ID NO. 61 29 CCCTAGATTCSEQ ID NO. 62 30 TCTTCGCTAC SEQ ID NO. 63 31 ACGCACCAGC SEQ ID NO. 64 32GCACCCAACC SEQ ID NO. 65 33 GTATCCAACG SEQ ID NO. 66 34 CCTTTAACGASEQ ID NO. 67 35 TCCTACGCTT SEQ ID NO. 68 36 ATGTGAGAAC SEQ ID NO. 69 37GGTATAACAG SEQ ID NO. 70 38 CTAAGACGAC SEQ ID NO. 71 39 ACTCACGATASEQ ID NO. 72 40 TAACCCTTTT SEQ ID NO. 73 41 CAATCCCACA SEQ ID NO. 74 42TAGTACATTC SEQ ID NO. 75 43 AACCCTAGCG SEQ ID NO. 76 44 GATCATCCTTSEQ ID NO. 77 45 AGCCAAGTAC SEQ ID NO. 78 46 TTCGACGACC SEQ ID NO. 79 47GCCATCCCTC SEQ ID NO. 80 48 CACTTACGGC SEQ ID NO. 81 49 CTTATGACATSEQ ID NO. 82 50 GCAAGCCTTC SEQ ID NO. 83 51 ACTCCTGCTT SEQ ID NO. 84 52TTACAATTAC SEQ ID NO. 85 53 ACTTGATGAC SEQ ID NO. 86 54 TCCGCCTTTTSEQ ID NO. 87 55 CGCTTAAGCT SEQ ID NO. 88 56 GGTGACATGC SEQ ID NO. 89 57TTCTTACTAG SEQ ID NO. 90 58 CGCCACTTTA SEQ ID NO. 91 59 GACATTACTTSEQ ID NO. 92 60 ACCGAGGCAC SEQ ID NO. 93 61 CGATAATCTT SEQ ID NO. 94 62ACCCTCACCT SEQ ID NO. 95 63 TCGAACCCGC SEQ ID NO. 96 64 GGTGTAGCACSEQ ID NO. 97 65 GCTTGATCCC SEQ ID NO. 98 66 ACATTACATC SEQ ID NO. 99 67CCCTAAGGAC SEQ ID NO. 100 68 TCGTCAATGC SEQ ID NO. 101 69 AAAGCATATCSEQ ID NO. 102 70 TCTGTAGGGC SEQ ID NO. 103 71 CGTTCCCTGT SEQ ID NO. 10472 GTATTCACTT SEQ ID NO. 105 73 ACGTCATTGC SEQ ID NO. 106 74 TCAGCGTCCTSEQ ID NO. 107 75 GCCCAGATAC SEQ ID NO. 108 76 CCTAAAACTT SEQ ID NO. 10977 AAGACCAGAT SEQ ID NO. 110 78 GATGATTGCC SEQ ID NO. 111 79 TAATTCTACTSEQ ID NO. 112 80 CACCGTAAAC SEQ ID NO. 113 81 AATGACGTTC SEQ ID NO. 11482 CTCCCTTCAC SEQ ID NO. 115 83 TACGCCATCC SEQ ID NO. 116 84 GTTCATCCGCSEQ ID NO. 117 85 AACGCTTTCC SEQ ID NO. 118 86 TCCTGGTACT SEQ ID NO. 11987 GCTTTGCTAT SEQ ID NO. 120 88 CATGATCAAC SEQ ID NO. 121 89 TAGACAGCCTSEQ ID NO. 122 90 AGTAGGTCAC SEQ ID NO. 123 91 CCCAATACGC SEQ ID NO. 12492 GTAATCCCTT SEQ ID NO. 125 93 GCATCGTAAC SEQ ID NO. 126 94 AAACACCCATSEQ ID NO. 127 95 TGCCGGACTC SEQ ID NO. 128 96 CTCTTCGATT SEQ ID NO. 129Circularization Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a circularizing step. Methods forcircularizing a polynucleotide fragment comprise: joining a linearpolynucleotide fragment at its first end to a first oligonucleotideadaptor, and joining the linear polynucleotide fragment at its secondend to a second oligonucleotide adaptor, and circularizing the resultinglinear construct. In some embodiments, the circularized construct caninclude nicks on opposite strands. In some embodiments, methods forcircularizing a polynucleotide fragment comprise joining each end of alinear polynucleotide fragment to a blocking oligonucleotide adaptorwhich comprise a double-stranded oligonucleotide adaptor (duplex) havingan overhang cohesive portion that anneals with a blockingoligonucleotide which can be a separate single-stranded oligonucleotide.In some embodiments, the blocking oligonucleotide adaptor comprises (i)a first and a second single-stranded oligonucleotide annealed togetherto form a duplex having an overhang portion and (ii) a thirdsingle-stranded oligonucleotide annealed to the overhang portion,wherein the overhang portion of the first and second double-strandedoligonucleotide adaptor are capable of annealing with each other, andwherein for the first and second double-stranded oligonucleotideadaptors the end of the duplex having the overhang portion forms anadaptor-joining end and the other end of the duplex forms atarget-joining end. In some embodiments, circularizing the linearconstruct includes removing the third single-stranded oligonucleotide(or a single-stranded portion of an oligonucleotide) from the overhangportions of the first and/or second oligonucleotide adaptors so as toexpose the overhang portions of the adaptor-joining ends. In someembodiments, the exposed overhang ends of two separate adaptors can beannealed together, thereby circularizing the linear construct. In someembodiments, the adaptors can hybridize without complete overlap betweenthe overhang ends, so as to leave a gap or nick on one or both strandsat the junction between the adaptor-joining ends of the first and secondadaptors (FIGS. 1A and B-3). In some embodiments, the circularizedconstruct can include nicks on opposite strands.

In some embodiments, a blocking oligonucleotide can be removed from anoverhang end, and a pair of overhang ends can anneal with each other,under conditions suitable for nucleic acid duplex annealing and/ordenaturing. One skilled in the art can conduct nucleic acid annealingand/or denaturation by modify temperature, salts, formamide, length oftime, and other factors. In some embodiments, the circularized constructcan include at least one nick in the adaptor-joining region on at leastone strand.

In some embodiments, any linear molecules remaining after acircularization step can be removed by treatment with an enzyme thatdegrades linear nucleic acids, including treatment with Plasmid Safe™ATP-Dependent DNase kit (Epicentre). In some embodiments, linear nucleicacids can be removed with a column or CsCl centrifugation.

Nick Translation Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a nick translation step. A nicktranslation reaction can be conducted at least once at any stage in thelibrary preparation workflow. A nick translation reaction can be used tomove a nick to a new position along a nucleic acid or to close a nick orgap. In some embodiments, a nick translation reaction can be a coupled5′ to 3′ DNA polymerization/degradation reaction, or a coupled 5′ to 3′DNA polymerization/strand displacement reaction. A nick translationreaction can be conducted with a DNA polymerase and deoxynucleotidetriphosphates. Methods for performing nick translation reactions areknown to those of skill in the art (Rigby, P. W. et al. (1977), J. Mol.Biol. 113, 237). Methods for preparing mate pair libraries using aninternal adaptor (IA) and a nick translation reaction are known (U.S.2009/0181861). A variety of suitable polymerases can be used to conducta nick translation reaction, including for example, E. coli DNApolymerase I, Taq DNA polymerase, Vent DNA polymerase, Klenow DNApolymerase I, Tfi DNA polymerase, Bst DNA polymerase, and phi29 DNApolymerase. Depending on the enzyme used, a nick translation reactioncan proceed by 5′ to 3′ exonuclease activity, or by 5′ to 3′ stranddisplacement. In some embodiments, a mutant enzyme with low activity canbe used to conduct a nick translation reaction. Mutant enzymes canexhibit lower extension rates, lower 5′ to 3′ exonuclease activity,lower 5′ to 3′ polymerase activity, lower 5′ to 3′ strand displacementactivity, or any combination thereof. In some embodiments, mutantenzymes can be sensitive to reaction conditions such as, for example,cations, temperature or pH.

The distance that a nick travels (moves) can be modulated by reactionconditions, such as reaction time, reaction temperature, the polymeraseused, pH, ions or cations present, and/or salt conditions. A nicktranslation reaction can be conducted at a temperature range of about 0°C. to about 40° C., or about 5° C. to about 10° C., or about 10° C. toabout 15° C., or about 15° C. to about 20° C., or about 20° C. to about25° C., or about 25° C. to about 30° C., or about 30° C. to about 35°C., or about 35° C. to about 40° C. A nick translation reaction can beconducted for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 minutes. A nick translation reaction can beterminated or slowed by increasing the temperature, decreasing thetemperature, altering the pH, altering the ions present, altering thesalt conditions, and/or addition of a chelating agent. In someembodiments, circularized nucleic acid molecule can be cleaved byallowing a nick translation reaction to proceed around the nucleic acidmolecule until it encounters the other nick; so as to self-cleave thecircular molecule.

Methods for Releasing a Mate Pair:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a step for releasing a linear matepair from a circular construct. In some embodiments, a linear mate pairconstruct can be released from a circular construct by employing anycombination of enzymatic reactions to (a) open a nick to make a gap(e.g., on a first strand) and (b) cleave the strand opposite the nick(e.g., on the opposite strand). In some embodiments, a nick can beopened to a gap with an exonuclease such as a T7 exonuclease, lambdaexonuclease, E. coli exonuclease III, DNase, or an ATP-dependent DNase.In some embodiments, a strand opposite the gap can be cleaved with asingle-strand specific endonuclease enzyme such as 51 nuclease, nucleaseP1, nuclease BAL-31 or mung bean nuclease. In some embodiments, acircular construct can be subjected to random shearing forces to releaselinear fragments and a linear mate pair construct (FIG. 5).

Methods for Purifying:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a purifying step. A purifying step caninclude separating polynucleotides fragments from non-desirablecomponents (e.g., buffers, salts, enzymes, primer-dimers, or excessadaptors or primers). In some embodiments, a purification procedure canbe conducted at least once at any stage in a workflow. Purificationprocedures include without limitation: bead purification, columnpurification, gel electrophoresis, dialysis, alcohol, precipitation,size-selective PEG precipitation and the like. In some embodiments, apolynucleotide fragment, joined to a blocking oligonucleotide adaptorhaving a binding partner (e.g., biotin), can be separated fromundesirable reagents in a reaction by binding the binding partner to asurface (e.g., planar or a bead) having a cognate binding partner (e.g.,streptavidin). In some embodiments, solid phase adherence/immobilizationmethods can be used for purification. For example, SPRI (Solid PhaseReversible Immobilization) beads from Agencourt can be used forpurification. In some embodiments, unreacted nucleic acids (e.g.,non-ligated blocking oligonucleotide adaptors, blockingoligonucleotides, polynucleotides of interest) can be removed bydigesting with DNase, leaving intact polynucleotides of interest joinedto blocking oligonucleotide adaptors.

Denaturation and Hybridization Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise a nucleic acid denaturation or anannealing step. Nucleic acids can be denatured or hybridized byadjusting the: temperature, pH, sodium concentration, and/or formamideconcentration. In some embodiments, the released linear mate pair can bedenatured into two single strands.

Immobilization Methods:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can comprise an immobilization step. A mate pair(single- or double-stranded) can be immobilized to a surface. In someembodiments, the surface can be a planar surface or a bead. For example,one end of a single-stranded mate pair can be modified with a chemicalcompound that will react with, and attach to another chemical compoundon the surface. In another example, one end of a single-stranded matepair can include a capture adaptor sequence for annealing to a captureprimer which is immobilized on a surface. In some embodiments, animmobilized single-stranded mate pair can be subjected to bridgeamplification reactions. In some embodiments, a mate pair construct canbe attached or immobilized to Ion Sphere™ Particles (sold as a componentof the Ion Xpress Template Kit (Part No. 4469001)) for clonalamplification Immobilization to Ion Sphere™ Particles can be performedessentially according to the protocols provided in the Ion Xpress™Template Kit v2.0 User Guide (Part No.: 4469004)).

Polymerases:

Provided herein are methods for preparing mate pair constructs and matepair libraries which can utilize one or more polymerases. In someembodiments, a polymerase includes any enzyme, or fragment or subunit ofthereof, that can catalyze polymerization of nucleotides and/ornucleotide analogs. In some embodiments, a polymerase requires theterminal 3′ OH of a nucleic acid primer to initiate nucleotidepolymerization. In some embodiments, a linker nucleic acid provides aterminal 3′OH for the polymerase to polymerize the nucleotides.

In some embodiments, nucleotide polymerization can occur in atemplate-dependent manner. In some embodiments, a polymerase can be ahigh fidelity polymerase. In some embodiments, a polymerase can be anaturally-occurring polymerase, recombinant polymerase, mutantpolymerase, variant polymerase, fusion or otherwise engineeredpolymerase, chemically modified polymerase, synthetic molecules, oranalog, derivative or fragment thereof.

In some embodiments, a mutant polymerase comprises substitution,insertion or deletion of one or more amino acids. In some embodiments, apolymerase can include two or more portions of polymerases linkedtogether. In some embodiments, a polymerase can be a fusion proteincomprising at least two portions linked to each other, where the firstportion comprises a peptide that can catalyze nucleotide polymerizationand a second portion comprising a second polypeptide. In someembodiments, a polymerase includes other enzymatic activities, such asfor example, 3′ to 5′ or 5′ to 3′ exonuclease activity, or stranddisplacement activity.

In some embodiments, a polymerase can be isolated from a cell, orgenerated using recombinant DNA technology or chemical synthesismethods. In some embodiments, a polymerase can be expressed inprokaryote, eukaryote, viral, or phage organisms. In some embodiments, apolymerase can be post-translationally modified proteins or fragmentsthereof.

In some embodiments, a polymerase can be a DNA polymerase and includewithout limitation bacterial DNA polymerases, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases.

In some embodiments, a polymerase can be a replicase, DNA-dependentpolymerase, primases, RNA-dependent polymerase (including RNA-dependentDNA polymerases such as, for example, reverse transcriptases), astrand-displacement polymerase, a thermo-labile polymerase, or athermo-stable polymerase. In some embodiments, a polymerase can be anyFamily A or B type polymerase. Many types of Family A (e.g., E. coli PolI), B (e.g., E. coli Pol II), C (e.g., E. coli Pol III), D (e.g.,Euryarchaeotic Pol II), X (e.g., human Pol beta), and Y (e.g., E. coliUmuC/DinB and eukaryotic RAD30/xeroderma pigmentosum variants)polymerases are described in Rothwell and Watsman 2005 Advances inProtein Chemistry 71:401-440. In some embodiments, a polymerase can be aT3, T5, T7, or SP6 RNA polymerase.

These and other polymerases are described by Rothwell and Watsman (2005Advances in Protein Chemistry 71:401-440). One skilled in the art willknow which polymerase(s) to select to conduct a polymerizing,amplifying, nick translating, and/or tailing reaction.

Nucleotides:

Provided herein are nucleotides which comprise mate pair constructs andmate pair libraries. Also provided herein are methods for preparing matepair constructs and mate pair libraries which can utilize one or moretypes of nucleotides. In some embodiments, a first and/or secondsingle-stranded oligonucleotide and/or any blocking oligonucleotide cancomprise nucleic acids having natural and/or analog nucleotides. In someembodiments, a nucleotide can be any compound that can bind selectivelyto, or can be polymerized by, a polymerase. In some embodiments,nucleotides can be polymerized by a polymerase in a primer extensionreaction. In some embodiments, a nucleotide can be a naturally-occurringnucleotide, or analog thereof. In some embodiments, a nucleotidecomprises a base, sugar and phosphate moieties. In some embodiments, anucleotide can lack a base, sugar or phosphate moiety. In someembodiments, a nucleotide can include a chain of phosphorus atomscomprising three, four, five, six, seven, eight, nine, ten or morephosphorus atoms. In some embodiments, a phosphorus chain can beattached to any carbon of a sugar ring, such as the 5′ carbon. In someembodiments, a phosphorus chain can be linked to the sugar with anintervening O or S. In some embodiments, one or more phosphorus atoms ina phosphorus chain can be part of a phosphate group having P and O. Insome embodiments, the phosphorus atoms in the chain can be linkedtogether with intervening O, NH, S, methylene, substituted methylene,ethylene, substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R(where R can be a 4-pyridine or 1-imidazole). In some embodiments, thephosphorus atoms in the chain can have a side group having O, BH₃, or S.In some embodiments, a phosphorus atom having a side group other than Ocan be a substituted phosphate group. In some embodiments, a nucleotidecan be attached to a label (e.g., reporter moiety). In some embodiments,a label can be a fluorophore. In some embodiments, a fluorophore can beattached to the terminal phosphate group (or substitute phosphategroup). In some embodiments, a nucleotide can comprise a non-oxygenmoiety (e.g., thio- or borano-moieties) that replaces an oxygen moietythat bridges the alpha phosphate and the sugar of the nucleotide, orbridges the alpha and beta phosphates of the nucleotide, or bridges thebeta and gamma phosphates of the nucleotide, or between any other twophosphates of the nucleotide, or any combination thereof. In someembodiments, nucleotides can be biotinylated.

In some embodiments, a nucleotide can be joined to a binding partner,such as biotin. For example, a biotin moiety can be joined to a base,sugar or any phosphate group of a nucleotide. Various biotinylatednucleotides are commercially-available (see Jena Bioscience, Germany).

In some embodiments, a nucleotide can be a ribonucleotide,deoxyribonucleotide, ribonucleotide polyphosphate, deoxyribonucleotidepolyphosphate, peptide nucleotides, metallonucleosides, phosphonatenucleosides, and modified phosphate-sugar backbone nucleotides, analogs,derivatives, variants or modified versions thereof.

Sequencing Methods:

Next Generation and Single Molecule Sequencing Methods:

Provided herein are mate pair constructs and mate pair libraries thatcan be sequenced using any sequencing technology, includingoligonucleotide probe ligation and detection (e.g., SOLiD™ from LifeTechnologies, WO 2006/084131), probe-anchor ligation sequencing (e.g.,Complete Genomics™ or Polonator™), sequencing-by-synthesis (e.g.,Genetic Analyzer and HiSeg™, from Illumina), pyrophosphate sequencing(e.g., Genome Sequencer FLX from 454 Life Sciences), ion-sensitive orsemiconductor sequencing (e.g., Personal Genome Machine from IonTorrent™ Systems, Life Technologies), or single molecule sequencingplatforms (e.g., HeliScope™ from Helicos™).

Ion-Sensitive or Semiconductor Sequencing Methods:

Provided herein are mate pair constructs and mate pair libraries thatcan be sequenced using methods that detect one or more byproducts ofnucleotide incorporation. The detection of polymerase extension bydetecting physicochemical byproducts of the extension reaction, caninclude pyrophosphate, hydrogen ion, charge transfer, heat, and thelike, as disclosed, for example, in Pourmand et al, Proc. Natl. Acad.Sci., 103: 6466-6470 (2006); Purushothaman et al., IEEE ISCAS,IV-169-172; Rothberg et al, U.S. Patent Publication No. 2009/0026082;Anderson et al, Sensors and Actuators B Chem., 129: 79-86 (2008); Sakataet al., Angew. Chem. 118:2283-2286 (2006); Esfandyapour et al., U.S.Patent Publication No. 2008/01666727; and Sakurai et al., Anal. Chem.64: 1996-1997 (1992).

Reactions involving the generation and detection of ions are widelyperformed. The use of direct ion detection methods to monitor theprogress of such reactions can simplify many current biological assays.For example, template-dependent nucleic acid synthesis by a polymerasecan be monitored by detecting hydrogen ions that are generated asnatural byproducts of nucleotide incorporations catalyzed by thepolymerase. Ion-sensitive sequencing (also referred to as “pH-based” or“ion-based” nucleic acid sequencing) exploits the direct detection ofionic byproducts, such as hydrogen ions, that are produced as abyproduct of nucleotide incorporation. In one exemplary system forion-based sequencing, the nucleic acid to be sequenced can be capturedin a microwell, and nucleotides can be floated across the well, one at atime, under nucleotide incorporation conditions. The polymeraseincorporates the appropriate nucleotide into the growing strand, and thehydrogen ion that is released can change the pH in the solution, whichcan be detected by an ion sensor. This technique does not requirelabeling of the nucleotides or expensive optical components, and allowsfor far more rapid completion of sequencing runs. Examples of suchion-based nucleic acid sequencing methods and platforms include the IonTorrent PGM™ sequencer (Life Technologies Corporation).

In some embodiments, one or more nucleic acid fragments produced usingthe methods, systems and kits of the present teachings can be used as asubstrate for a biological or chemical reaction that is detected and/ormonitored by a sensor including a field-effect transistor (FET). Invarious embodiments the FET is a chemFET or an ISFET. A “chemFET” orchemical field-effect transistor, is a type of field effect transistorthat acts as a chemical sensor. It is the structural analog of a MOSFETtransistor, where the charge on the gate electrode is applied by achemical process. An “ISFET” or ion-sensitive field-effect transistor,is used for measuring ion concentrations in solution; when the ionconcentration (such as H+) changes, the current through the transistorwill change accordingly. A detailed theory of operation of an ISFET isgiven in “Thirty years of ISFETOLOGY: what happened in the past 30 yearsand what may happen in the next 30 years,” P. Bergveld, Sens. Actuators,88 (2003), pp. 1-20.

In some embodiments, the FET may be a FET array. As used herein, an“array” is a planar arrangement of elements such as sensors or wells.The array may be one or two dimensional. A one dimensional array can bean array having one column (or row) of elements in the first dimensionand a plurality of columns (or rows) in the second dimension. The numberof columns (or rows) in the first and second dimensions may or may notbe the same. The FET or array can comprise 102, 103, 104, 105, 106, 107or more FETs.

In some embodiments, one or more microfluidic structures can befabricated above the FET sensor array to provide for containment and/orconfinement of a biological or chemical reaction. For example, in oneimplementation, the microfluidic structure(s) can be configured as oneor more wells (or microwells, or reaction chambers, or reaction wells,as the terms are used interchangeably herein) disposed above one or moresensors of the array, such that the one or more sensors over which agiven well is disposed detect and measure analyte presence, level,and/or concentration in the given well. In some embodiments, there canbe a 1:1 correspondence of FET sensors and reaction wells.

Microwells or reaction chambers are typically hollows or wells havingwell-defined shapes and volumes which can be manufactured into asubstrate and can be fabricated using conventional microfabricationtechniques, e.g. as disclosed in the following references: Doering andNishi, Editors, Handbook of Semiconductor Manufacturing Technology,Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMSand Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al,Silicon Micromachining (Cambridge University Press, 2004); and the like.Examples of configurations (e.g. spacing, shape and volumes) ofmicrowells or reaction chambers are disclosed in Rothberg et al, U.S.patent publication 2009/0127589; Rothberg et al, U.K. patent applicationGB24611127.

In some embodiments, the biological or chemical reaction can beperformed in a solution or a reaction chamber that is in contact with orcapacitively coupled to a FET such as a chemFET or an ISFET. The FET (orchemFET or ISFET) and/or reaction chamber can be an array of FETs orreaction chambers, respectively.

In some embodiments, a biological or chemical reaction can be carriedout in a two-dimensional array of reaction chambers, wherein eachreaction chamber can be coupled to a FET, and each reaction chamber isno greater than 10 μm³ (i.e., 1 pL) in volume. In some embodiments eachreaction chamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pLin volume. A reaction chamber can optionally be 22, 32, 42, 52, 62, 72,82, 92, or 102 square microns in cross-sectional area at the top.Preferably, the array has at least 102, 103, 104, 105, 106, 107, 108,109, or more reaction chambers. In some embodiments, the reactionchambers can be capacitively coupled to the FETs.

FET arrays as used in various embodiments according to the disclosurecan be fabricated according to conventional CMOS fabricationstechniques, as well as modified CMOS fabrication techniques and othersemiconductor fabrication techniques beyond those conventionallyemployed in CMOS fabrication. Additionally, various lithographytechniques can be employed as part of an array fabrication process.

Exemplary FET arrays suitable for use in the disclosed methods, as wellas microwells and attendant fluidics, and methods for manufacturingthem, are disclosed, for example, in U.S. Patent Publication No.20100301398; U.S. Patent Publication No. 20100300895; U.S. PatentPublication No. 20100300559; U.S. Patent Publication No. 20100197507,U.S. Patent Publication No. 20100137143; U.S. Patent Publication No.20090127589; and U.S. Patent Publication No. 20090026082, which areincorporated by reference in their entireties. Examples of an array caninclude Ion Torrent™ System arrays, such as the 314™, 316™ and 318™Chips (Life Technologies) used in conjunction with an Ion Torrent™ PGMSequencer (Life Technologies, Part No. 4462917).

In one aspect, the disclosed methods, compositions, systems, apparatusesand kits can be used for carrying out label-free nucleic acidsequencing, and in particular, ion-based nucleic acid sequencing. Theconcept of label-free detection of nucleotide incorporation has beendescribed in the literature, including the following references that areincorporated by reference: Rothberg et al, U.S. patent publication2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470(2006). Briefly, in nucleic acid sequencing applications, nucleotideincorporations are determined by measuring natural byproducts ofpolymerase-catalyzed extension reactions, including hydrogen ions,polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase).

In some embodiments, the disclosure relates generally to methods forpreparing mate pair constructs or mate pair libraries that can besequenced using hydrogen ion-sensitive sequencing methods. In oneexemplary embodiment, the disclosure relates generally to a method forpreparing mate pair constructs or mate pair libraries, comprising: (a)providing a first and a second double-stranded oligonucleotide adaptoreach having (i) a first and a second single-stranded oligonucleotideannealed together to form a duplex having an overhang portion and (ii) athird single-stranded oligonucleotide annealed to the overhang portion,wherein the overhang portion of the first and second double-strandedoligonucleotide adaptors are capable of annealing with each other, andwherein for the first and a second double-stranded oligonucleotideadaptors the end of the duplex having the overhang portion forms anadaptor-joining end and the other end of the duplex forms atarget-joining end; (b) joining the target-joining end of the firstdouble-stranded oligonucleotide adaptor to a first end of the lineardouble-stranded polynucleotide of interest; (c) joining thetarget-joining end of the second double-stranded oligonucleotide adaptorto a second end of the linear double-stranded polynucleotide ofinterest; (d) removing the third single-stranded oligonucleotide fromthe first and the second double-stranded oligonucleotide adaptors so asto expose the overhang portions of the adaptor-joining ends; (e)annealing the overhang portions of the adaptor-joining ends therebyforming a circular polynucleotide of interest, wherein the circularpolynucleotide of interest comprises at least one nick at a junctionbetween the adaptor-joining ends of the first and the seconddouble-stranded oligonucleotide adaptors; (f) performing a nicktranslation reaction on the at least first nick to move the nick to anew position within the polynucleotide of interest; (g) performing anexonuclease reaction so as to remove at least a portion of the strandhaving the nick, so as to open the nick into a gap; and (h) cleaving thestrand opposite the gap so as to release a linear mate pair construct.

In some embodiments, the mate pair constructs or mate pair library canbe sequenced using an ion-sensitive sequencing method. In someembodiments, the sequencing method is performed by incorporating one ormore nucleotides in a template-dependent fashion into a newlysynthesized nucleic acid strand.

Optionally, the methods can further include producing one or more ionicbyproducts of such nucleotide incorporation.

In some embodiments, the methods can further include detecting theincorporation of the one or more nucleotides into the sequencing primer.Optionally, the detecting can include detecting the release of hydrogenions.

In another embodiment, the disclosure relates generally to a method forsequencing a nucleic acid, comprising: (a) producing mate pairconstructs or mate pair libraries according to the methods disclosedherein; (b) disposing a plurality of mate pair constructs or mate pairlibraries into a plurality of reaction chambers, wherein one or more ofthe reaction chambers are in contact with a field effect transistor(FET). Optionally, the method further includes contacting at least oneof the mate pair constructs disposed into one of the reaction chamberswith a polymerase, thereby synthesizing a new nucleic acid strand bysequentially incorporating one or more nucleotides into a nucleic acidmolecule. Optionally, the method further includes generating one or morehydrogen ions as a byproduct of such nucleotide incorporation.Optionally, the method further includes detecting the incorporation ofthe one or more nucleotides by detecting the generation of the one ormore hydrogen ions using the FET.

In some embodiments, the detecting includes detecting a change involtage and/or current at the at least one FET within the array inresponse to the generation of the one or more hydrogen ions.

In some embodiments, the FET can be selected from the group consistingof: ion-sensitive FET (isFET) and chemically-sensitive FET (chemFET).

One exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™ sequencer(Life Technologies), which is an ion-based sequencing system thatsequences nucleic acid templates by detecting hydrogen ions produced asa byproduct of nucleotide incorporation. Typically, hydrogen ions arereleased as byproducts of nucleotide incorporations occurring duringtemplate-dependent nucleic acid synthesis by a polymerase. The IonTorrent PGM™ sequencer detects the nucleotide incorporations bydetecting the hydrogen ion byproducts of the nucleotide incorporations.The Ion Torrent PGM™ sequencer can include a plurality of nucleic acidtemplates to be sequenced, each template disposed within a respectivesequencing reaction well in an array. The wells of the array can each becoupled to at least one ion sensor that can detect the release of H⁺ions or changes in solution pH produced as a byproduct of nucleotideincorporation. The ion sensor comprises a field effect transistor (FET)coupled to an ion-sensitive detection layer that can sense the presenceof H⁺ ions or changes in solution pH. The ion sensor can provide outputsignals indicative of nucleotide incorporation which can be representedas voltage changes whose magnitude correlates with the H⁺ ionconcentration in a respective well or reaction chamber. Differentnucleotide types can be flowed serially into the reaction chamber, andcan be incorporated by the polymerase into an extending primer (orpolymerization site) in an order determined by the sequence of thetemplate. Each nucleotide incorporation can be accompanied by therelease of H⁺ ions in the reaction well, along with a concomitant changein the localized pH. The release of H⁺ ions can be registered by the FETof the sensor, which produces signals indicating the occurrence of thenucleotide incorporation. Nucleotides that are not incorporated during aparticular nucleotide flow may not produce signals. The amplitude of thesignals from the FET can also be correlated with the number ofnucleotides of a particular type incorporated into the extending nucleicacid molecule thereby permitting homopolymer regions to be resolved.Thus, during a run of the sequencer multiple nucleotide flows into thereaction chamber along with incorporation monitoring across amultiplicity of wells or reaction chambers can permit the instrument toresolve the sequence of many nucleic acid templates simultaneously.Further details regarding the compositions, design and operation of theIon Torrent PGM™ sequencer can be found, for example, in U.S. patentapplication Ser. No. 12/002,781, now published as U.S. PatentPublication No. 2009/0026082; U.S. patent application Ser. No.12/474,897, now published as U.S. Patent Publication No. 2010/0137143;and U.S. patent application Ser. No. 12/492,844, now published as U.S.Patent Publication No. 2010/0282617, all of which applications areincorporated by reference herein in their entireties.

In some embodiments, the disclosure relates generally to use of matepair constructs or mate pair libraries produced using any of themethods, systems and kits of the present disclosure in methods ofion-based sequencing. Use of such mate pair constructs or mate pairlibraries in ion-based sequencing reactions can be advantageous becausethe methods of the disclosure permit isolation of polynucleotides (e.g.,tags) of a desired size that can be selected to match the read lengthcapacity of the ion-based sequencing system.

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations can be detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzedextension reactions. In one embodiment, templates each having a primerand polymerase operably bound can be loaded into reaction chambers (suchas the microwells disclosed in Rothberg et al, cited herein), afterwhich repeated cycles of nucleotide addition and washing can be carriedout. In some embodiments, such templates can be attached as clonalpopulations to a solid support, such as a particle, bead, or the like,and said clonal populations are loaded into reaction chambers. As usedherein, “operably bound” means that a primer is annealed to a templateso that the primer's 3′ end may be extended by a polymerase and that apolymerase is bound to such primer-template duplex, or in closeproximity thereof so that binding and/or extension takes place whenevernucleotides are added.

In each addition step of the cycle, the polymerase can extend the primerby incorporating added nucleotide only if the next base in the templateis the complement of the added nucleotide. If there is one complementarybase, there is one incorporation, if two, there are two incorporations,if three, there are three incorporations, and so on. With each suchincorporation there is a hydrogen ion released, and collectively apopulation of templates releasing hydrogen ions changes the local pH ofthe reaction chamber. The production of hydrogen ions is monotonicallyrelated to the number of contiguous complementary bases in the template(as well as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereare a number of contiguous identical complementary bases in the template(i.e. a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, can be proportional tothe number of contiguous identical complementary bases. If the next basein the template is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, an additional stepcan be performed, in which an unbuffered wash solution at apredetermined pH is used to remove the nucleotide of the previous stepin order to prevent misincorporations in later cycles. In someembodiments, the after each step of adding a nucleotide, an additionalstep can be performed wherein the reaction chambers are treated with anucleotide-destroying agent, such as apyrase, to eliminate any residualnucleotides remaining in the chamber, which may result in spuriousextensions in subsequent cycles.

In one exemplary embodiment, different kinds of nucleotides are addedsequentially to the reaction chambers, so that each reaction can beexposed to the different nucleotides one at a time. For example,nucleotides can be added in the following sequence: dATP, dCTP, dGTP,dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposure followed bya wash step. The cycles may be repeated for 50 times, 100 times, 200times, 300 times, 400 times, 500 times, 750 times, or more, depending onthe length of sequence information desired.

In some embodiments, sequencing can be performed with an Ion Torrent™PGM™ sequencer. For example, mate pair constructs prepared as disclosedherein can be clonally amplified on Ion Sphere™ Particles as part of theIon Xpress™ Template Kit (Life Technologies Part No. 4469001). Templatepreparation can be performed essentially accordingly to the protocolsprovided in the Ion Xpress™ Template Kit v2.0 User Guide (LifeTechnologies, Part No. 4469004). The amplified DNA can then be sequencedon the Ion PGM™ sequencer (Ion Torrent™, Life Technologies, Part No.4462917) essentially according to the protocols provided in the IonSequencing Kit v2.0 User Guide (Ion Torrent™, Life Technologies, PartNo. 4469714) and using the reagents provided in the Ion Sequencing Kit(Ion Torrent™, Life Technologies, Part No. 4468997) and the Ion 314™Chip Kit (Ion Torrent™, Life Technologies, Part No. 4462923).

Systems:

Provided herein are systems comprising a blocking oligonucleotideadaptors which include (a) a first single-stranded oligonucleotideannealed to a second single-stranded oligonucleotide to form a duplexhaving an overhang portion and (b) a third single-strandedoligonucleotide (blocking oligonucleotide) annealed to the overhangportion, where the end of the duplex having the overhang portion forms aadaptor-joining end and the other end of the duplex forms atarget-joining end. The overhang portion can be exposed by removal ofthe third single-stranded oligonucleotide. In some embodiments, systemsfurther comprise one or more polynucleotides of interest for joining toone or more blocking oligonucleotide adaptors. In some embodiments, afirst and/or second single-stranded oligonucleotide can include abinding partner moiety (e.g., biotin). In some embodiments, the overhangportion of the first and second blocking oligonucleotide adaptors can becapable of annealing with each other. In some embodiments, a systemcomprises a pair of blocking oligonucleotide adaptors.

Provided herein are systems, comprising linear polynucleotide constructshaving a linear polynucleotide of interest joined at one or both endswith a blocking oligonucleotide adaptor. Blocking oligonucleotideadaptors can comprise: (a) a first single-stranded oligonucleotideannealed to a second single-stranded oligonucleotide to form a duplexhaving an overhang portion and (b) a third single-strandedoligonucleotide (blocking oligonucleotide) annealed to the overhangportion, where the end of the duplex having the overhang portion forms aadaptor-joining end and the other end of the duplex forms atarget-joining end. In some embodiments, the overhang portion of thefirst and second blocking oligonucleotide adaptors can be capable ofannealing with each other. In some embodiments, the thirdsingle-stranded oligonucleotide (blocking oligonucleotide) of the firstand second blocking oligonucleotide adaptors can be removed to exposethe overhang portions. In some embodiments, a first and/or secondsingle-stranded oligonucleotide can include a binding partner moiety(e.g., biotin).

Provided herein are systems, comprising linear polynucleotides havingeach end joined to a blocking oligonucleotide adaptor and circularized.For example, a circular construct includes a linear polynucleotide ofinterest joined at a first end with a target-joining end of a firstblocking oligonucleotide adaptor and joined at a second end with atarget-joining end of a second blocking oligonucleotide adaptor. Thefirst and second blocking oligonucleotide adaptors comprise: (a) a firstsingle-stranded oligonucleotide annealed to a second single-strandedoligonucleotide to form a duplex having an overhang portion and (b) athird single-stranded oligonucleotide (blocking oligonucleotide)annealed to the overhang portion, where the end of the duplex having theoverhang portion forms a adaptor-joining end and the other end of theduplex forms a target-joining end. In some embodiments, the overhangportion of the first and second blocking oligonucleotide adaptors can becapable of annealing with each other. In some embodiments, the thirdsingle-stranded oligonucleotide (or a single-stranded portion of anoligonucleotide) can be removed from the overhang portions of the firstand second oligonucleotide adaptors so as to expose the overhangportions of the adaptor-joining ends. In some embodiments, the exposedoverhang ends of two blocking oligonucleotide adaptors can be annealedtogether, thereby joining together the adaptor-joining ends of the firstand second blocking oligonucleotide adaptors so as to generate acircularized construct. In some embodiments, one or both polynucleotidestrands at the junction between the adaptor-joining ends of the firstand second adaptors can include a nick or gap. In some embodiments, afirst and/or second single-stranded oligonucleotide can include abinding partner moiety (e.g., biotin).

Kits

Provided herein are kits comprising reagents for conducting methods forjoining together one or more ends of polynucleotides of interest toblocking oligonucleotide adaptors, for preparing mate pair constructsand/or for preparing mate pair libraries having blocking oligonucleotideadaptors. In some embodiments, kits can include any combination of: oneor more types of blocking oligonucleotide adaptors (with or withoutbiotin) (FIGS. 1A and B-7); reagents for fragmenting a polynucleotide ofinterest; reagents for end-repairing the ends of the fragments of apolynucleotide of interest; reagents for size-selecting nucleic acids;reagents for joining one or both ends of a fragment of a polynucleotideof interest to one or more types of adaptors (e.g., blockingoligonucleotide adaptors or additional types of adaptors); reagents forcircularizing a linear polynucleotide of interest joined to one or moreblocking oligonucleotide adaptors; reagents for performing a nicktranslation reaction or exonuclease digestion reaction or strandextension reaction; reagents for releasing a linear mate pair construct;reagents to perform a tailing reaction; reagents for purifying apolynucleotide of interest (e.g., as a mate pair construct); reagentsfor quantifying nucleic acids; reagents for amplifying nucleic acidsand/or reagents for sequencing nucleic acids. In some embodiments, thekits can include a set of instructions and genome assembly guides can beincluded. Such material can be, for example, in print or in digitalform. In some embodiments, the kits can include any combination of:various enzymes to conduct reactions such as ligating, end-repairing,size-selecting, adaptor-joining, circularizing, nick-translating,degrading linear nucleic acids; releasing a mate-pair, tailing, andamplifying; beads for nucleic acid capture; reagents for washing;reagents for PCR amplification; adaptors (e.g., P1, P2, A, blockingoligonucleotide adaptors); PCR primers; nucleic acid purificationcolumns; and/or components for nucleic acid gel extraction. In someembodiments, the kits include one or more adaptors having identificationsequences (e.g., barcodes).

Embodiments of the present teachings can be further understood in lightof the following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1

Provided herein is one method for preparing the DNA to be ligated to theblocking oligonucleotide adaptors.

5 ug of genomic DNA was added to a final volume of 125 ul in H₂O or TE.The DNA was sheared via Hydroshear. After shearing, the DNAconcentration was confirmed with QUBIT. The expected recovery is about75%. The DNA was purified with a PURELINK column. Use the B2 buffer with55% isopropanol. The DNA was eluted in 50 ul of E1. The DNA was loadedand eluted a second time. The DNA yield was about 3.2 ug as measured byQUIBIT. The DNA was size-selected on a 1% agarose gel. The DNA wasextracted with a QUICK GEL EXTRACTION KIT. The gel slice was about 0.6ml. The DNA was extracted using 2 QUICK GEL EXTRACTION COLUMNS, elutedin 50 ul of E5 per column. For a 2-3 kb insert library, the DNA yieldwas about 1.3 ug as measured by QUIBIT.

The DNA was end-polished: DNA in 100 ul; 30 ul 5× End polishing buffer;3.75 ul of 10 mM dNTPs; 4.5 ul of End polishing enzyme 1; 12 ul Endpolishing enzyme 2; incubated at room temperature for 30 minutes.

The DNA was purified with a PURELINK column, using B2 buffer with 55%isopropanol. The DNA was eluted 50 ul of E1. The DNA was loaded andeluted a second time. The DNA yield was about 1.2 ug as measured byQUIBIT. The DNA is now ready for ligating to the blockingoligonucleotide adaptors.

Example 2

Provided herein are methods for circularizing a polynucleotide ofinterest, and constructing a long mate pair library using blockingoligonucleotide adaptors having non-palindrome sequences.

Shearing DNA

1 Kb-3 Kb of DNA was sheared using the COVAIS S2 System or HydroshearDNA Shearing Device. A speed-vac was used to reduce the volume for thesize selection procedure. The sheared DNA was size-selected on a 1%agarose gel prepared in 1×TAE buffer with 10 μL of 1:10,000 SYBR SAFEgel stain per 100 mL volume.

Size-Selecting the DNA

Gel slices containing the desired DNA was cut from the gel. The gelslices were weighed in either 2 mL (gel mass of 400 mg or less) or 15 mL(gel mass from 400 mg to 3,750 mg) tubes, depending on the estimatedvolume. Assumption: every mg of gel mass equals 1 μL of volume whendissolved. This assumption was used to add 3× volumes of GelSolubilization Buffer (L3) (Invitrogen, catalog # K2100-12) to the tubecontaining the gel slices. The gel slices were dissolved byshaking/vortexing the tube at room temperature until the gel slicesdissolved completely (˜15 minutes). Dissolving the gel slice attemperatures higher than room temperature (e.g., 50° C.) will denaturethe DNA and cause heteroduplex formation. The gel slices were dissolvedin 1-gel volume of isopropanol. For example, 10 μL of isopropanol wasadded to 10 mg of gel and mixed well. The dissolved gel mixture wasapplied to a Quick Gel Extraction column(s) in WashTube(s) (Invitrogen,catalog # K2100-12). One column was used per 400 mg agarose or less than□2000 μL of dissolved gel mixture was loaded per column. More columnswere used when necessary. The column was centrifuged at >12,000 xG for 1minute, and the flow-though was discarded. The column was placed back onthe Wash Tube(s). 500 μL of Wash Buffer (W1) with ethanol was flowedthrough the Quick Gel Extraction column(s). The columns were centrifugedat >12,000 xG for 1 minute, and the flow-through was discarded. TheQuick Gel Extraction columns were centrifuged again at maximum speed for2 minutes to remove any residual Wash Buffer. The Quick Gel Extractioncolumns were transferred to clean 1.5-mL LOBIND tubes (Eppendorf,catalog #0030 108.035). The DNA was eluted from the Quick Gel Extractioncolumns with 50 μL of Elution Buffer (E1). The columns were rested for 1minute at room temperature. For large fragments, the time was increasedto 10 minutes for increased yield. The DNA was eluted from the column bycentrifugation at >12,000 xG for 1 minute. The eluate in the 1.5-mLLOBIND contained the purified DNA. The eluate was flowed through QuickGel Extraction columns, then rested for 1 minute at room temperature.The columns were centrifuge at >12,000×g for 1 minute. The DNA yield wasquantitated using QUIBIT (Invitrogen, catalog # Q32861).

End-Repair the Size Selected DNA

The size-selected DNA was mixed in a LOBIND 1.5 mL tube or a PCR tube aslisted in Table 3. The mixture was incubated at room temperature (20 to25° C.) for 30 minutes, and heat-killed at 75° C. for 30 minutes, andiced.

TABLE 3 Component Concentration Volume Water 17.0 T4 DNA Ligase Buffer5X 20 dNTP 10 mM 4.0 T4 Polynucleotide Kinase 10 U/uL 4 T4 DNAPolymerase 5 U/uL 5 Post-SS DNA 50 Total Reaction Volume (ul) 100Pre-Assembly of the Blocking Oligonucleotide Adaptors

The right (R) and left (L) adaptors were biotinylated and included thefollowing sequences:

Adaptor L:

Adaptor R:

100 ul of 25 uM of the left/right adaptor was prepared by mixing thefirst and second single-stranded oligonucleotides, and the blockingoligonucleotide as follows:

-   -   20 ul first ss-oligonucleotde (125 uM)    -   30 ul third ss-oligonucleotide (125 uM)    -   20 ul second ss-oligonucleotide (125 uM)    -   10 ul water    -   20 ul 5× ligase buffer

A 100 ul of 25 uM of the left/right adaptor was prepared by mixing thefirst and second single-stranded oligonucleotides and the blockingoligonucleotide as follows:

-   -   20 ul first ss-oligonucleotide (125 uM)    -   30 ul third ss-oligonucleotide (125 um)    -   20 ul second ss-oligonucleotide (125 um)    -   10 ul water    -   20 ul 5× ligase buffer

Annealing was conducted in a thermocycler.

Ligating Insert DNA to the Adaptors

The amount of adaptor needed for the ligation reaction was calculatedbased on the amount of the DNA from the End Repair step.X pmol/ug DNA=1 ug DNA×[(10⁶ pg)/(1 ug)]×[(1 pmol)/(660 pg)]×[1/(averageinsert size)].Y ul adapter needed=# ug DNA×[(X pmol)/(1 ug DNA)]×100×[(1 ul adaptorneeded)/(50 pmol)].For example:For 12 ug of purified end-repaired DNA with an average insert size of1.5 kb

X  p mol/ug  DNA = 1  ug  DNA × [10⁶  pg/1  ug] × [1  p mol/660  pg] × [1/1500] = 1.0  p mol/ug  DNA$\mspace{79mu}{\begin{matrix}{{Y\mspace{14mu}{uL}\mspace{14mu}{adaptor}\mspace{14mu}{needed}} = {12\mspace{14mu}{ug}\mspace{14mu}{DNA} \times \left\lbrack {1.0\mspace{14mu} p\;{mol}\text{/}1\mspace{14mu}{ug}\mspace{14mu}{DNA}} \right\rbrack \times}} \\{100 \times \left\lbrack {1\mspace{14mu}{uL}\mspace{14mu}{adaptor}\mspace{14mu}{needed}\text{/}50\mspace{14mu} p\;{mol}} \right\rbrack} \\{= {24\mspace{14mu}{uL}\mspace{14mu}{adaptor}\mspace{14mu}{needed}}}\end{matrix}\quad}$

The ligation reaction was set up as shown in Table 4 below. The ligationreaction was incubated at room temperature (20 to 25° C.) for 30minutes.

TABLE 4 Component Concentration Volume End-Polished DNA 100 T4 DNALigase Buffer 5X 10 ATP 25 mM 6 Adaptor L 25 uM x Adaptor R 25 uM x T4DNA Ligase 5X 7.5 Water x Total Reaction Volume (ul) 150AMPURE XP Bead Purifying the Ligation Products

One volume of nuclease-free water was added to the sample reaction. 0.8volumes of AMPURE XP bead mix (Agencourt, catalog # A63880) was addedper original volume of DNA sample. The mixture was vortexed andincubated at room temperature for 5 minutes. The tubes were placed on aDYNALMAG rack for 1 minute. The tubes were kept on the magnetic rack.The supernatant was discarded. 600 ul of 70% ethanol was added to thetubes. The tubes were pulse-vortexed thoroughly and pulse spin. Thetubes were again placed on a DYNALMAG rack for 1 minute. The supernatantwas discarded. Again 600 ul of 70% ethanol was added to the tubes. Thetubes were pulse-vortexed thoroughly and pulse spin. The tubes wereremoved the DYNALMAG rack (Invitrogen) and pulse spin the in amicrofuge. The residual supernatant was discarded. The beads wereallowed to dry for 3 minutes. The tubes were removed from the DYNALMAGrack and 50 ul of E1 Buffer (Applied Biosystems) was added to the tubes.The tubes were vortexed and incubated at room temperature for 3 minutesor longer. The tubes were placed on the DYNALMAG for 1 minute. The E1eluate was transferred to a new 1.5 LOBIND tube. 2 pL of the DNA wasquantitated by QUIBIT (Invitrogen, catalog # Q32861).

Circularizing the DNA Via Intra Molecular Hybridization

The amount of DNA in the hybridization reaction was calculated to be 0.5ng/ul. 10× Plasmid-Safe buffer was diluted to 1× in water. Thehybridization reaction was set up as shown in Table 5 below.

TABLE 5 Componet Volume DNA Volume (uL) 50 Plasmid-Safe 1X Buffer xTotal Volume = 0.5 ng/ul of DNA in mix x

The hybridization reaction was incubated in a heat block at 70° C. for 5min, and placed on ice for 30 minutes (typically no more than 1 ml pertube). Large volumes were aliquoted into PCR tubes, 100 ul per tube. Thesamples were pooled together after cooling.

In an alternative method, the circularization step was conducted inNick-Translation Buffer. The annealing reaction was incubated in a heatblock at 70° C. for 5 min, then placed at 5° C. for 5-30 minutes. At thesame time, the DNA pol I and dNTP were incubated at 5° C. Thepre-chilled DNA pol I and dNTP were added into circularization mix atthe end of incubation. B2-S buffer was added to stop the reaction.

Isolating the Circularized DNA

The circularized DNA was treated with Plasmid-Safe ATP Dependent DNase(Epicentre Biotechnologies) and 25 mM ATP as shown in Table 6 below. Themixture was incubated at 37° C. for 40 minutes.

TABLE 6 Component Volume Epicentre 25 mMATP total vol/1000*40 EpicentrePlasmid-Safe Dnase x/150Purifying the DNA with SOLID Library Micro Column Purification Kit

Empty PURELINK PCR Micro columns (Invitrogen) were pre-spun incollection tubes at 10,000 xG for one minute. 4 volumes of PURELINKBinding Buffer (B2-S) with isopropanol was added to 1 volume of sampleand mixed well. One PURELING PCR Micro column was used per 4-5 mL ofsample in Binding Buffer (B2).

≤700 μL of sample in Binding Buffer (B2) was loaded onto the PURELINKMicro columns in collection tubes. The columns were centrifuged at10,000×xG for 15 seconds except for the last loading. The flow-throughwas discarded after each spin. After the last loading, the column wasspun spin 1 minute. The dsDNA bound to the column. These steps wererepeated until the entire sample was loaded onto the columns.

The PURELINK Micro columns were placed back into the same collectiontube. 650 μL of Wash Buffer (W1) with ethanol was used to wash thecolumn. The columns were centrifuged at 10,000×xG for 1 minute. Theflow-through was discarded. The centrifugation was repeated at 14,000×xGto remove residual wash buffer. The columns were transferred to clean1.5-mL LOBIND tubes. 25 μL of Elution Buffer (E1) was loaded onto to thecenter of the column to elute the DNA, then the columns were allowed tostand for 1 minute at room temperature. The columns were spun at 14,000xG for 1 minute. The eluate was loaded back onto the column(s), then letthe column and allowed to stand for 1 minute at room temperature. Thecolumns were spun at 14,000 xG for 1 minute.

In an alternative method, the circularized DNA was purified using AMPUREbeads. 0.3× volume of AMPURE beads and 0.7× volume of Bead Buffer (14%PEG8000, 2.5M NaCl) were added to the PLASMIDSAFE DNase-treatedcircularized DNA. Standard bead capture, wash and elution steps wereconducted.

Nick Translating the Circularized DNA

A 0.2-mL LOBIND tube was prepared as shown in Table 7 below.

TABLE 7 Component Volume DNA Volume After Qubit (uL) 25 Nuclease-FreeWater (uL) 57 Nick-Translation Buffer 10 10 mM dNTPs (uL) 5 Total Volume(uL) 97

The mixture was pre-incubated without DNA polymerase I at 5° C. in athermocycler for more than 2 minutes. 3 μL of DNA polymerase I was addedto a separate 0.2 mL tube, then pulse-spun. The DNA polymerase I waspre-incubated at 5° C. in a thermocycler for at least 1 minute.

The reaction mix was added to the tube containing the DNA polymerase Iand thoroughly mixed. The nick translation reaction was performed usingthe “no heated lid” feature on the thermocycler for 12 minutes.

400 μL of PURELINK Binding Buffer (B2-S) with isopropanol was added to a1.5-mL LOBIND tube. At the end of the 12 minutes, the nick translationreaction was immediately transferred to the 1.5-mL LOBIND tubecontaining Binding Buffer (B2) to denature the enzyme and stop thereaction. The nick-translated DNA was purified using a SOLID LibraryMicro Column Purification Kit.

T7 Exonuclease Digestion

A T7 exonuclease reaction was set up as shown in Table 8 below. Thereaction was incubated at 37° C. for 15 minutes. The enzyme was heatinactivated at 70° C. for 20 minutes. The reaction was chilled on icefor 5 minutes.

TABLE 8 Components Volume DNA 25 10X Buffer 4 5 T7 Exonuclease 10 U/uL 2Water 18 Total Volume (ul) 50S1 Nuclease Digestion

1 μL of S1 Nuclease was freshly diluted to 25 U/μL with S1 NucleaseDilution Buffer. The S1 nuclease reaction was set up as shown in Table 9below. The reaction was incubated at 37° C. for 30 minutes.

TABLE 9 Components Volume DNA 50 3M NaCl 1.67 S1 Nuclease 25 U/uL 3Total Volume (ul) 54.7Alternative DNase and T7 Exonuclease Digestion

The nick-translated DNA was resuspended in PLASMIDSAFE buffer, ATP andPLASMIDSAFE DNase was added to digest the linear DNA fragments (37° C.for 30 minutes). At the end of incubation 20 U of T7 exonuclease wasadded directly into the reaction mixture and incubate for another 5minutes. The enzymes were heat inactivated at 70° C. for 20 minutes. Thereaction was chilled on ice for 5 minutes.

1 μL of S1 Nuclease was freshly diluted to 25 U/μL with S1 NucleaseDilution Buffer. The S1 nuclease reaction was set up as shown in Table 9above. The reaction was incubated at 37° C. for 30 minutes.

A-Tailing Reaction

The A-tailing reaction was set up as shown in Table 10 below.

TABLE 10 Component Concentration Volume React 2 Buffer 10X 10 dATP 10 mM4 dNTPs 10 mM 1 Ambion Klenow Exo- 5 U/uL 6 Water 29 T7/S1-Treated DNA50 Total Volume (ul) 100

The reaction was incubated at 37° C. for 30 minutes.

The reaction was stopped by mixing compounds as shown in Table 11 below.

TABLE 11 Component Volume A-tailed DNA 100 0.5M EDTA 5 Bead BindingBuffer 200 Nuclease-free Water 95 Total (ul) 400DNA Binding to Streptavidin Beads

A 1×BSA solution was prepared. The tube of DYNABEADS MYONE StreptavidinC1 was vortexed, then 50 μL of the beads was transferred into a 1.5-mLLOBIND Tube. 300 μL of Bead Wash Buffer was added to the 90 μL ofsolution of beads, the beads were vortexed for 15 seconds, thenpulse-spun. The tube was placed on a DYNALMAG rack for 1 minute. Afterthe solution appeared clear the supernatant was discarded. 300 μL of1×BSA was added to the tube and vortexed for 15 seconds, thenpulse-spun. The tube was placed on a DYNALMAG rack for 1 minute. Afterthe solution appeared clear the supernatant was discarded. 300 μL ofBead Binding Buffer was added, and the beads were vortexed for 15seconds, then pulse spun. The tube was placed on a DYNALMAG rack for 1minute. After the solution appeared clear the supernatant was discarded.The entire 400 μL of solution of library DNA in Bead Binding Buffer wasadded to the pre-washed streptavidin beads, then vortexed. The solutionwas rotated at room temperature (20 to 25° C.) for 30 minutes, thenpulse spun.

Washing the Bead-DNA Complex

1× Ligase Buffer (total volume=400 ul) was prepared. The tube ofbead-DNA complex was placed on a magnetic rack for at least 1 minute.After the solution appeared clear, the supernatant was discarded. Thebead-DNA were resuspended in 300 μL of Bead Wash Buffer, thentransferred to a new the beads to a new 1.5-mL LOBIND tube. The bead-DNAwas vortexed 15 seconds, then pulse-spun. The tube of bead-DNA complexwas placed on a magnetic rack for at least 1 minute. After the solutionappeared clear, the supernatant was discarded. The bead-DNA wasresuspended in 300 μL of Bead Wash Buffer. The bead-DNA was vortexed for15 seconds, then pulse-spun. The tube of bead-DNA complex was placed ona magnetic rack for at least 1 minute. After the solution appearedclear, the supernatant was discarded. The bead-DNA was resuspended in300 μL of Bead Wash Buffer. The bead-DNA was vortexed for 15 seconds,then pulse-spun. The tube of bead-DNA complex was placed on a magneticrack for at least 1 minute. After the solution appeared clear, thesupernatant was discarded. The bead-DNA was resuspended in 300 μL of 1×Ligase Buffer. The bead-DNA was vortexed for 15 seconds, thenpulse-spun. The tube of bead-DNA complex was placed on a magnetic rackfor at least 1 minute. After the solution appeared clear, thesupernatant was discarded. The bead-DNA was resuspended in 93 μL of 1×Ligase Buffer.

Ligating the P1 and P2 Adaptors to the DNA

The bead-DNA was prepared for ligation to P1 and P2 adaptors as shown inTable12 below:

TABLE 12 Component Volume DNA-bead complex 93 P1 Adaptor (ds), 50 μM 1P2 Adaptor (ds), 50 μM 1 T4 DNA Ligase, 5 U/μL 5 Total Volume (μL) 100

The ligation reaction was incubated at room temperature (20 to 25° C.)on a rotator for 20 minutes. The tube of bead-DNA-P1/P2 complex wasplaced on a magnetic rack for at least 1 minute. After the solutionappeared clear, the supernatant was discarded. The beads wereresuspended in 300 μL of Bead Wash Buffer and transferred to a new1.5-mL LOBIND tube. The beads were vortexed for 15 seconds, thenpulse-spun. The tube of bead-DNA-P1/P2 complex was placed on a magneticrack for at least 1 minute. After the solution appeared clear, thesupernatant was discarded. The bead-DNA-P1/P2 complex was resuspended in300 μL of Bead Wash Buffer. The beads were vortexed for 15 seconds, thenpulse-spun. The tube of bead-DNA-P1/P2 complex was placed on a magneticrack for at least 1 minute. After the solution appeared clear, thesupernatant was discarded. The beads were resuspended in 300 μL ofElution Buffer (E1). The tube of bead-DNA-P1/P2 complex was placed on amagnetic rack for at least 1 minute. After the solution appeared clear,the supernatant was discarded. The beads were resuspended in 30 μL ofElution Buffer (E1).

PCR Amplifying the Paired Tag Library

The paired tag library was amplified with a PLATINUM PCR AmplificationMix (Invitrogen). The PLATINUM PCR amplification mix contains aproofreading enzyme for high fidelity amplification. The PCR reactionwas set up as shown in Table 13 below:

TABLE 13 Component Volume Platinum ® PCR Amplification Mx‡ 70 LibraryPCR Primer 1, 50 μM 1.4 Library PCR Primer 2, 50 μM 1.4 Total (ul) 72.8

The master mix was vortexed. For a negative control, 23 μL of the PCRmaster mix was added to a PCR tube. 4 μL of DNA-bead complex solutionwas added to the remaining 49.8 μL of PCR master mix, vortexed, thendivided evenly (˜25 μL) between two PCR tubes (tubes #1 and 2). Tube #1:10 cycles, tube #2 and negative control tube: 14 cycles. Thethermocycler settings are shown in Table 14 below.

TABLE 14 Stage Step Temp Time Holding Nick Translation 72° C. 20 minHolding Denature 94° C. 3 min Cycling Denature 94° C. 15 sec Anneal 62°C. 15 sec Extend 70° C. 1 min Holding Extend 70° C. 5 min Holding  4° C.?

Amplification was confirmed on a pre-case 2% E-GEL EX (Invitrogen). Thesize of amplified library should be between 275 and 325 bp, but 250 to350 bp is acceptable. Detailed methods for preparing a mate pairconstruct and/or mate pair library can be found in “Mate-Pair LibraryPreparation; 5500 Series SOLiD™ Systems” (from Applied Biosystems,publication part No. 4460958, hereby incorporated by reference in itsentirety).

Example 3

This Example illustrates another method of making a paired tag library.

In this Example, a nucleic acid sequence is fragmented to produce aplurality of polynucleotide fragments. The ends of the polynucleotidefragments are end repaired to form blunt-ended polynucleotide fragments.The polynucleotide fragments are then subjected to a size selectionmethod to generate polynucleotides of interest that are the desired sizefor example, about 10 kb in size. Adaptors are joined to the ends ofeach size-selected polynucleotide of interest, thereby forming aplurality of adaptor modified polynucleotides of interest. The adaptormodified polynucleotides of interest are ligated to produce a pluralityof circular nucleic acid molecules, wherein a nick is introduced betweenthe adaptor and the polynucleotide of interest. A nick translationreaction is performed on the circular nucleic acid molecules to producea plurality of circular nucleic acid molecules each having at least onenick present within its corresponding polynucleotide fragment. Theresulting circular nucleic acid molecules are cleaved at a pointopposite a nick to release the paired tags, thereby producing a pairedtag library. The paired tag clones can be clonally amplified by, forexample, by emulsion PCR, and subsequently sequenced by, for example,Ion Torrent™ PGM Sequencing.

Example 4

This Example illustrates a paired tag library preparation workflow.

In this Example, a nucleic acid sequence is fragmented to form randomlength polynucleotides. The random length polynucleotides are subjectedto a size selection method to remove smaller and/or largerpolynucleotides outside the desired length of the polynucleotide ofinterest. The size selected polynucleotides of interest are modified tohave the appropriate end configurations to match one or more adaptorsthat are ligated to the polynucleotides of interest to form adaptormodified polynucleotides of interest. After ligation, the circularizedadaptor modified polynucleotides contain one or more nicks located closeto the polynucleotide of interest. The one or more nicks are thenextended into the polynucleotide of interest by nick translation. Aftertranslation of the nicks, the circular population is then cut at the oneor more nick positions, thereby releasing the paired tag, and therebyproducing a paired tag library. The paired tag library can be clonallyamplified by, for example, by emulsion PCR, and subsequently sequencedby, for example, Ion Torrent™ PGM Sequencing.

Example 5

This example provides another method for the preparation of a longmate-pair library.

Shearing DNA

DNA (1-5 μg DNA for 3 kb insert library or 1-20 μg DNA for 10 kb insertlibrary) was sheared using a HydroShear® DNA Shearing Device (DigiLab,MA) according to the manufacturer's instructions. For 3 kb inserts, DNAwas sheared using the HydroShear® standard shearing assembly with aspeed code of 13 for 20 cycles. For 10 kb inserts, DNA was sheared usingthe HydroShear® large shearing assembly with a speed code of 10 for 20cycles. The final volume of sheared DNA was reduced using a SpeedVac® orconcentrator to obtain about 70 μl of sheared DNA for a 3 kb insertlibrary and less than 100 μl for a 10 kb insert library.

End-Repairing the Sheared DNA

The sheared DNA was mixed in a LoBind 1.5 mL tube or a PCR tube aslisted in Table 15. The mixture was incubated at room temperature (20 to25° C.) for 30 minutes.

TABLE 15 Component 3 kb library 10 kb library Sheared DNA ~65 ul  <100ul   5x Reaction Buffer 20 ul  30 ul  10 mM dNTP 4 ul 6 ul End PolishingE1 4 ul 6 ul End Polishing E2 5 ul 8 ul Nuclease-free water VariesVaries Total 100 ul  150 ul 

The following reagents were added to the end repaired DNA solutions:

Component 3-kb Library 10-kb library 20% SDS 8.5 ul 13 ul 10XBlueJuice ™ Gel  10 ul 16 ul Loading Buffer

The end-repaired DNA was denatured for 10 minutes at 65° C. The sampleswere placed on ice for 5 minutes and loaded onto wells on an appropriatepercent agarose gel in 1×TAE buffer with 20 μL of 10,000×SYBR® Safe gelstain per 200 mL volume. Typically, a 0.6% gel can be used for a 10-kblibrary and a 1% agarose gel can be used to size select a 3-kb library.

Size-Selecting the DNA

The size-selection step was conducted using a SOLiD™ Library Quick GelExtraction Kit (Invitrogen Part No. 4443711). Gel slices containing thedesired DNA was cut from the gel. For a 3 kb insert library, the gelband was removed from 2.8 kb-3.5 kb from a 1% agarose gel. For a 10 kbinsert library, the band was removed from the 10 kb-11 kb size rangefrom a 0.6% agarose gel. The gel slices were weighed in either 1.5 mLLobind tube (gel mass of 200 mg or less) or 15 mL (gel mass greater than200 mg) tubes, depending on the estimated volume. A 30 μL volume of GelSolubilization Buffer (L3) was added for every 10 mg of gel mass to thetube containing the gel slices. The gel slices were dissolved byshaking/vortexing the tube at room temperature until the gel slicesdissolved completely (˜15 minutes). Dissolving the gel slice attemperatures higher than room temperature (e.g., 50° C.) will denaturethe DNA and cause heteroduplex formation. The gel slices were dissolvedin 1-gel volume of isopropanol. For example, 10 μL of isopropanol wasadded to 10 mg of gel and mixed well. The dissolved gel mixture wasapplied to a Quick Gel Extraction column(s). One column was used per 400mg agarose or less than □2000 μL of dissolved gel mixture. The columnwas centrifuged at >12,000 xG for 1 minute, and the flow-though wasdiscarded. 500 μL of Wash Buffer (W1) with ethanol was flowed throughthe Quick Gel Extraction column(s). The columns were centrifugedat >12,000 xG for 1 minute, and the flow-through was discarded. TheQuick Gel Extraction columns were centrifuged again at maximum speed for2 minutes to remove any residual Wash Buffer. The Quick Gel Extractioncolumns were transferred to clean 1.5-mL LOBIND tubes (Eppendorf,Catalog No. 0030 108.035). The DNA was eluted from the Quick GelExtraction columns with 50 μL of Elution Buffer (E5). The columns wererested for 10 minutes at room temperature. The DNA was eluted from thecolumn by centrifugation at >12,000 xG for 1 minute. If more than onecolumn was used, the eluate was pooled and reduced in volume with aSpeedVac® or concentrator to a volume less than 60 μl to performligation of the mate pair adaptors to the DNA. The DNA yield wasquantitated using the QUBIT™ dsDNA HS assay kits (Invitrogen, CatalogNo. Q32851) and the Qubit® 2.0 (Invitrogen, Part No. Q32866).

Pre-Assembly of the Blocking Oligonucleotide Adaptors

In this example, left (L) and right (R) adaptors included biotin andincluded the following sequences. However, in an alternative methodnon-biotinylated adaptors can be used as blocking adaptors.

Adaptor L:

Adaptor R:

100 μl of 25 μM of the left/right adaptor was prepared by mixing thefirst and second single-stranded oligonucleotides, and the blockingoligonucleotide as follows:

-   -   20 μl first ss-oligonucleotde (125 μM)    -   30 μl third ss-oligonucleotide (125 μM)    -   20 μl second ss-oligonucleotide (125 μM)    -   10 μl water    -   20 μl 5× ligase buffer

Annealing of the blocking adaptors was conducted in a thermocycler. Inan alternative method, the left and right adaptors were notpre-assembled and ligated to the size-selected, end-repaired DNA asdescribed in the following step.

Ligating DNA to the Adaptors

The ligation step adds the mate pair adaptors to the sheared,end-repaired DNA. The mate pair adaptors are lacking a 5′ phosphate atthe non-joining end; as a result, there is a nick on each strand whenthe DNA is circularized. The amount of adaptor needed for the ligationreaction was calculated based on the amount of the DNA from the sizeselection step.ug to pmol conversion factor=(10⁶ pg)/(1 ug)×(1 pmol)/(660pg)×1/(average insert size).Y ul adapter needed=# ug DNA×(ug to pmol conversion factor)×50×(1 uladaptor needed)/(25 pmol).For example, 1 ug of purified size-selected DNA with an average insertsize of about 3 kb:ug to pmol conversion factor=(10⁶ pg)/(1 ug)×(1 pmol)/(660pg)×1/(3000)=0.5 pmol/μg DNAY ul adapter needed=1 ug DNA×(0.5 pmol)/(ug DNA)×50×(1 ul adaptorneeded)/(25 pmol)=1 μl adaptor needed.

The ligation reaction was set up as shown in Table 16 below. Theligation reaction was incubated at room temperature (20 to 25° C.) for30 minutes.

TABLE 16 End-repaired DNA <60 ul 5x Reaction Buffer 20 ul MPR Adaptor(ds), 25 uM Y ul MPL Adaptor (ds), 25 um Y ul T4 DNA ligase, 5 U/ul 10ul Nuclease-free water Varies Total 100 ulAMPure® XP Bead Purifying the Ligation Products

1.5× volume of nuclease-free water was added to the sample reaction. 1.6volumes of AMPure® XP bead mix (Agencourt, Catalog No. A63880) was addedper original volume of DNA sample. The mixture was vortexed for 15seconds, pulse-spin and incubated at room temperature for 5 minutes. Thetubes were placed on a DYNALMAG rack for 1 minute until the solutioncleared. The supernatant was discarded. 600 ul of freshly prepared 70%ethanol was added to the tube. The tubes were kept on a DYNALMAG rackfor at least 1 minute and then the supernatant was discarded. Again 600ul of freshly prepared 70% ethanol was added to the tubes, left to standfor 1 minute and the residual supernatant was discarded. The tubes werepulse spin, returned to the rack and any residual supernatant wasdiscarded. The beads were allowed to dry for 3 minutes. The tubes wereremoved from the DYNALMAG rack and 50 ul of E1 Buffer was added to thetubes. The tubes were vortexed and incubated at room temperature for 3minutes. The tubes were placed on the DYNALMAG for 1 minute until thesolution cleared. The E1 eluate was transferred to a new 1.5 LOBINDtube. 1 μL of the DNA sample was quantitated by QUBIT™ dsDNA HS assaykits (Invitrogen, Catalog No. Q32851) and the Qubit® 2.0 Fluorometer(Invitrogen, Part No. Q32866.

Circularizing the DNA Via Intra Molecular Hybridization

The amount of DNA in the hybridization reaction was calculated to afinal concentration of 0.5 ng/μl. For example, the total volume of thecircularization reaction (T, ul) was calculated for a knownconcentration of DNA (DNA ng/ul) and a known volume of DNA (V). Forexample, if the DNA concentration is 5 ng/ul and the V=50 ul, then T=500ul.

The hybridization reaction was set up as shown in Table 17 below.

TABLE 17 Component Volume DNA V ul 10x Plasmid-Safe ™ T/10 ul BufferNuclease-free water T-(T/10)-V ul Total T ul

The hybridization reaction was incubated in a heat block at 70° C. for 5min, and placed on ice. Large volumes were aliquoted into PCR tubes, 100ul per tube. The samples were pooled together after cooling.

Isolating the Circularized DNA

The circularized DNA was treated with Plasmid-Safe DNase to eliminateuncircularized DNA (Epicentre Biotechnologies) and 100 mM ATP as shownin Table 18 below. The mixture was incubated at 37° C. for 40 minutes.

TABLE 18 Component Volume DNA T ul ATP, 100 mM T/100 ul Plasmid-Safe ™DNase, T/100 ul 10 u/ul Total T ulBased on the above, if T=800 μl, then ATP (100 mM) is 8 μl andPlasmid-Safe DNase (10 u/μl) is 8 μl.Purifying the Circularized DNA with AMPure® XP Beads

The bead suspension reaction was set up as follows:

Sample reaction=T μl

Bead dilution buffer=0.7×T μl

Agencourt AMPure® XP Reagent=0.3×T μl

The mixture was vortexed for 15 seconds, pulse-spin and incubated atroom temperature for 5 minutes. The tubes were placed on a DYNALMAG rackfor 1 minute until the solution cleared. The supernatant was discarded.600 ul of freshly prepared 70% ethanol was added to the tube. The tubeswere kept on a DYNALMAG rack for at least 1 minute and then thesupernatant was discarded. Again 600 ul of freshly prepared 70% ethanolwas added to the tubes, left to stand for 1 minute and the residualsupernatant was discarded. The tubes were pulse spin, returned to therack and any residual supernatant was discarded. The beads were allowedto dry for 3 minutes. The tubes containing the dried sample wereresuspended in 94 μl of a pre-mixed solution (containing 84 μlnuclease-free water and 10 μl of Nick Translation Buffer (E1)). Thetubes were gently vortexed for 15 seconds, pulse-spin and incubated atroom temperature for 3 minutes. The tubes were placed on the DYNALMAGfor 1 minute until the solution cleared. The supernatant was transferredto a new 1.5 LOBIND tube. Optionally, 1 μL of the DNA sample wasquantitated by QUBIT™ dsDNA HS assay kits (Invitrogen, Catalog No.Q32851) and the Qubit® 2.0 Fluorometer (Invitrogen, Part No. Q32866).

Nick Translating the Circularized DNA

Nick translation using E. coli DNA polymerase I translates the nick intothe genomic DNA region. The size of the mate-paired tags to be producedcan be controlled by adjusting the reaction time and temperature.

A 0.2-mL LOBIND tube was prepared as shown in Table 19 below.

TABLE 19 Component Volume DNase treated, ~90 ul purified DNA 10 mM dNTP 5 ul

The mixture was pre-incubated without DNA polymerase I at 5° C. in athermocycler for 2-3 minutes. 5 μL of DNA polymerase I was added to aseparate 0.2 mL tube, then pulse-spun. The DNA polymerase I waspre-incubated at 5° C. in a thermocycler for at least 1 minute.

The reaction mix was added to the tube containing the DNA polymerase Iand thoroughly mixed. The nick translation reaction was performed usingthe “no heated lid” feature on the thermocycler for 10 minutes.

400 μL of PURELINK Binding Buffer (B2-S) with isopropanol was added to a1.5-mL LOBIND tube. At the end of the incubation, the nick translationreaction was immediately transferred to the 1.5-mL LOBIND tubecontaining Binding Buffer (B2-S) to denature the enzyme and stop thereaction. The nick-translated DNA was purified using a SOLiD LibraryMicro Column Purification Kit (Life Technologies) essentially accordingto the manufacturer's instructions. The purified DNA in elution buffer(E1) was stored at 4° C. or directly digested with T7 Exonuclease and S1nuclease.

T7 Exonuclease Digestion

T7 exonuclease recognizes nicks within the circularized DNA. The 5′-3′exonuclease activity digests the unligated strand away from themate-pair tags creating a gap in the sequence. This gap creates anexposed single-stranded region that is more easily recognized by S1nuclease. A T7 exonuclease reaction was set up as shown in Table 20below. The reaction was incubated at 37° C. for 15 minutes. The enzymewas heat inactivated at 70° C. for 20 minutes. The reaction was chilledon ice for 5 minutes.

TABLE 20 Components Volume DNA 25 10X Buffer 4 5 T7 Exonuclease 10 U/uL2 Water 18 Total Volume (ul) 50

S1 Nuclease Digestion

1 μL of S1 Nuclease was freshly diluted to 50 U/μL with S1 NucleaseDilution Buffer. The S1 nuclease reaction was set up as shown in Table21 below. The reaction was incubated at 37° C. for 45 minutes.

TABLE 21 Components Volume DNA 50 3M NaCl 1.7 S1 Nuclease 50 U/uL 2Total Volume (ul) 53.7

The digested DNA was then purified using the Agencourt AMPure® XPReagent. The bead suspension reaction was set up as follows:

Sample reaction=53 μl

Agencourt AMPure® XP Reagent=95 μl

Total=148 μl.

The mixture was vortexed for 15 seconds, pulse-spin and incubated atroom temperature for 5 minutes. The tubes were placed on a DYNALMAG rackfor 1 minute until the solution cleared. The supernatant was discarded.600 ul of freshly prepared 70% ethanol was added to the tube. The tubeswere kept on a DYNALMAG rack for at least 1 minute and then thesupernatant was discarded. Again 600 ul of freshly prepared 70% ethanolwas added to the tubes, left to stand for 1 minute and the residualsupernatant was discarded. The tubes were pulse spin, returned to therack and any residual supernatant was discarded. The beads were allowedto dry for 3 minutes. The tubes were removed from the DYNALMAG and 50 μlof Elution buffer (E1) was added to each tube. The mixture was vortexedfor 15 seconds, pulse-spin and incubated at room temperature for atleast 3 minutes. The tubes were placed on a DYNALMAG rack for at least 1minute until the solution cleared. The supernatant was transferred to anew 1.5 LOBIND tube.

Alternative Method for DNase and T7 Exonuclease Digestion

The nick-translated DNA was resuspended in PLASMIDSAFE buffer, ATP andPLASMIDSAFE DNase was added to digest the linear DNA fragments (37° C.for 30 minutes). At the end of incubation 20 U of T7 exonuclease wasadded directly into the reaction mixture and incubate for another 5minutes. The enzymes were heat inactivated at 70° C. for 20 minutes. Thereaction was chilled on ice for 5 minutes.

1 μL of S1 Nuclease was freshly diluted to 25 U/μL with S1 NucleaseDilution Buffer. The S1 nuclease reaction was set up as shown in Table21 above. The reaction was incubated at 37° C. for 30 minutes

At this point, the reaction mixture contains the linear mate pairlibrary. Various other steps can be included by the user to furthermodify the linear mate pair library such as: adding a dA-tail to thedigested DNA, binding the library molecules to streptavidin beads,ligating P1, P2 or A adaptors to the DNA, nick-translating theamplifying the mate pair library, or evaluating the library for exampleusing a High Sensitivity DNA Chip (Agilent). The above additional stepscan be performed for example as outlined in Example 6 of thisapplication. In one embodiment, the evaluated mate pair library can beclonally amplified using an Ion Xpress™ Template Kit (Life TechnologiesPart No. 4469001) essentially accordingly to the protocols provided inthe Ion Xpress™ Template Kit v2.0 User Guide (Life Technologies, PartNo. 4469004), hereby incorporated by reference in their entirety. Theamplified DNA can then be sequenced on the Ion PGM™ sequencer (IonTorrent™, Life Technologies, Part No. 4462917) essentially according tothe protocols provided in the Ion Sequencing Kit v2.0 User Guide (IonTorrent™, Life Technologies, Part No. 4469714), hereby incorporated byreference in its entirety, and using the reagents provided in the IonSequencing Kit (Ion Torrent™, Life Technologies, Part No. 4468997) andthe Ion 314™ Chip Kit (Ion Torrent™, Life Technologies, Part No.4462923), both of which are hereby incorporated in their entirety.

Example 6

The linear mate pair library prepared according to Example 5 can befurther processed to end-repair the T7/S1 digested DNA. An end-repairreaction was set up as shown in Table 22 below. The reaction wasincubated at room temperature for 30 minutes.

TABLE 22 T7/S1 digested DNA 50 ul 5x Reaction Buffer 20 ul 10 mM dNTPmix  2 ul End Polishing Enzyme 2  2 ul Nuclease-free water 26 ul Total100 ul 

After incubation, 5.0 μl of 0.5 M EDTA was added to stop the reaction.

The sample volume (105 μl of stopped end-repair mix) was added to 200 μlbead binding buffer and with 95 μl nuclease-free water to bring thetotal volume to 400 μl.

DNA Binding to Streptavidin Beads

Dynabeads® MyOne™ Streptavidin C1 specifically binds to thebiotin-labeled mate pair adaptor in the library molecules to purify thelibrary from side products. The beads were pre-washed with a 1×BSAsolution.

The tube of DYNABEADS MYONE Streptavidin C1 was vortexed, then 50 μL ofthe beads was transferred into a 1.5-mL LOBIND Tube. 500 μL of Bead WashBuffer was added to the 50 μL of solution of beads, the beads werevortexed for 15 seconds, then pulse-spun. The tube was placed on aDYNALMAG rack for 1 minute. After the solution appeared clear thesupernatant was discarded. 500 μL of 1×BSA was added to the tube andvortexed for 15 seconds, then pulse-spun. The tube was placed on aDYNALMAG rack for 1 minute. After the solution appeared clear thesupernatant was discarded. 500 μL of Bead Binding Buffer was added, andthe beads were vortexed for 15 seconds, then pulse spun. The tube wasplaced on a DYNALMAG rack for 1 minute. After the solution appearedclear the supernatant was discarded. The entire 400 μL of samplecontaining the library DNA in Bead Binding Buffer was added to thepre-washed streptavidin beads, then vortexed for 15 seconds. Thesolution was rotated at room temperature (20 to 25° C.) for 30 minutes,then pulse spun.

Washing the Bead-DNA Complex

1× Reaction Buffer (total volume=600 μl) was prepared as follows: 5×Reaction Buffer=120 μl and Nuclease-free water 480 μl. The tube ofbead-DNA complex was placed on a magnetic rack for at least 1 minute.After the solution appeared clear, the supernatant was discarded. Thebead-DNA was washed three times as follows: 500 μL of Bead Wash Bufferwas added to the bead-DNA tube and placed on a magnetic rack for atleast 1 minute. After the solution appeared clear, the supernatant wasdiscarded. The bead-DNA was resuspended in 500 μL of 1× Reaction Buffer.The bead-DNA was vortexed for 15 seconds, then pulse-spun. The tube ofbead-DNA complex was placed on a magnetic rack for at least 1 minute.After the solution appeared clear, the supernatant was discarded. Thebead-DNA was resuspended in 87 μL of 1× Reaction Buffer.

Ligating Ion Adaptors to the DNA Library

The ligated library molecules are bound to streptavidin beads, washedand purified from ligation by-products. The bead-DNA is now ready forligation to adaptors, such as primer, sequencing or other functionalityadaptors. In this example, the bead-DNA was prepared for ligation to IonTorrent™ sequencing specific adaptors (Life Technologies). Thus, thebead-DNA was prepared for ligation to P1 Adaptors Ion (top and bottom)and A Adaptors Ion (top and bottom) as shown in Table 23 below:

TABLE 23 Component Volume DNA-bead complex 87 P1 Adaptor Ion (ds) 1.5 AAdaptor Ion (ds) 1.5 T4 DNA Ligase, 5 U/μL 10 Total Volume (μL) 100

The ligation reaction was incubated at room temperature (20 to 25° C.)on a rotator for 30 minutes. The tube of bead-DNA-P1/A complex wasplaced on a magnetic rack for at least 1 minute. After the solutionappeared clear, the supernatant was discarded. The beads wereresuspended in 500 μL of Bead Wash Buffer, vortexed for 15 seconds, thenpulse-spun. The tube of bead-DNA-P1/A complex was placed on a magneticrack for at least 1 minute. After the solution appeared clear, thesupernatant was discarded. The bead-DNA-P1/A complex was resuspended in500 μL of Bead Wash Buffer, vortexed for 15 seconds, then pulse-spun.The tube of bead-DNA-P1/A complex was placed on a magnetic rack for atleast 1 minute. After the solution appeared clear, the supernatant wasdiscarded. The beads were resuspended in 500 μL of Bead Wash Buffer,vortexed for 15 seconds, then pulse-spun. The tube of bead-DNA-P1/Acomplex was placed on a magnetic rack for at least 1 minute. After thesolution appeared clear, the supernatant was discarded. The beads wereresuspended in 500 μL of Elution Buffer (E1). The tube of bead-DNA-P1/Acomplex was placed on a magnetic rack for at least 1 minute. After thesolution appeared clear, the supernatant was discarded. The beads wereresuspended in 30 μL of Elution Buffer (E1).

PCR Amplifying the Paired Tag Library

The paired tag library was amplified with a Platinum® PCR AmplificationMix (Invitrogen). The Platinum® PCR amplification mix contains aproofreading enzyme for high fidelity amplification and nicktranslation. Before conducting the final nick-translation andamplification step, a trial amplification of the PCR primer andPlatinum® PCR Amplification Mix was performed to determine the optimumnumber of PCR cycles for each insert. PCR Primer 1 and PCR Primer 3 werepre-mixed at a final concentration of 5 μM in 10 mM Tris pH 7.5. Theprimer mix was then referred to as the Ion Library Amplification PrimerMix. A PCR reaction was set up as shown in Table 24 below:

TABLE 24 Component Volume Platinum ® PCR 70 Amplification Mix IonLibrary Amplification 2.5 Primer Mix Total Volume (μL) 72.5

The master mix was vortexed. For a negative control, ˜23 μL of the PCRmaster mix was added to a PCR tube (PCR #0). 4 μL of DNA-bead complexsolution was added to the remaining 50 μL of PCR master mix, vortexed,then divided evenly (˜25 μL) between two PCR tubes (tubes PCR #1 and 2).Each tube was subjected to thermocycling under the following conditions:

No. of Cycles No. of Cycles Sample No. for 3 kb insert for 10 kb insert0 16 20 1 12 16 2 16 20

The thermocycler settings are provided in Table 25 below.

TABLE 25 Stage Step Temp Time Holding Nick Translation 72° C. 20 minHolding Denature 94° C.  5 min Cycling Denature 94° C. 15 sec Anneal 58°C. 15 sec Extend 68° C.  1 min Holding —  4° C. variable

Amplification was confirmed using a pre-case 2% E-GEL EX GEL(Invitrogen). The presence of amplification product in the gel was usedas an indicator to select the optimal number of cycles foramplification.

The paired tag library was amplified after determining the optimalthermocycling conditions (procedure outlined above). PCR Primer 1 andPCR Primer 3 were pre-mixed at a final concentration of 5 μM each. Theprimer mix was then referred to as the Ion Library Amplification PrimerMix. A PCR amplification reaction master mix was set up as shown inTable 26 below:

TABLE 26 Component Volume Platinum ® PCR 200 Amplification Mix IonLibrary Amplification 10 Primer Mix Total Volume (μL) 210

The DNA-bead complex solution was placed on a magnetic rack for at least1 minute. After the solution appeared clear, the supernatant wasdiscarded being careful not to disturb the beads. The beads wereresuspended in PCR master mix of Table 26 above, vortexed for 15seconds, and loaded with the following settings into the thermocycler:

TABLE 27 Stage Step Temp Time Holding Nick Translation 72° C. 20 minHolding Denature 94° C.  5 min Cycling Denature 94° C. 15 sec Anneal 58°C. 15 sec Extend 68° C.  1 min Holding —  4° C. variablePurifying the DNA with SOLiD Library Micro Column Purification Kit

The amplified DNA was purified using SOLiD Library Micro ColumnPurification Kit. Empty PURELINK PCR Micro columns (Invitrogen) werepre-spun in collection tubes at 10,000 xG for one minute. 4 volumes ofBinding Buffer (B2-L) with isopropanol was added to 1 volume of sampleand mixed well.

The entire PCR sample was loaded onto the PURELINK Micro column in acollection tube. The column was centrifuged at 10,000×xG for 1 minute atroom temperature; the flow-through was discarded. The dsDNA is now boundto the column. The PURELINK Micro column was washed by adding 650 μL ofWash Buffer (W1) with ethanol to the column. The column was centrifugedat 10,000×xG for 1 minute at room temperature. The flow-through wasdiscarded. The centrifugation was repeated at 14,000×xG to removeresidual wash buffer. The column was transferred to a clean 1.5-mLLOBIND tube. 25 μL of Elution Buffer (E1) was loaded onto to the centerof the column to elute the DNA. The column was allowed to stand for 1minute at room temperature. The column was spun at 14,000 xG for 1minute. The eluate was loaded back onto the column, allowing the columnto stand for 1 minute at room temperature. The column was spun at 14,000xG for 1 minute at room temperature.

Size Select the Library with a SOLiD Library Size Selection Gel

A size-selection step was performed to ensure a library of distinctsize; however this step reduces the overall library yield. Thesize-selection step was conducted using a SOLiD™ Library Size SelectionGel. The library DNA sample was loaded onto a SOLiD Library SizeSelection Gel (2%) and run according to the manufacturer's instructions.The gel was run until the library product entered the collection well.The collection well was flushed with 20 μl of nuclease free water.

Check the Size Distribution of the Library

1 μl of the eluted library sample was analyzed using the AgilentTechnologies 2100 Bioanalyzer™ to ensure the library was of the expectedsize distribution. The library was quantitated to determine the librarydilution that results in a concentration within the optimized targetrange for Template Preparation (e.g., PCR-mediated addition of librarymolecules onto Ion Sphere™ Particles). The library is typicallyquantitated using an Ion Library Quantitation Kit (qPCR) (LifeTechnologies, Part No. 4468802) or Bioanalyzer™ (Agilent Technologies,Agilent 2100 Bioanalyzer) to determine the molar concentration of thelibrary, from which the Template Dilution Factor is calculated. Forexample, instructions to determine the Template Dilution Factor byquantitative real-time PCR (qPCR) can be found in the Ion LibraryQuantitation Kit User Guide (Life Technologies, Part No. 4468986),hereby incorporated by reference in its entirety.

While the principles of the present teachings have been described inconnection with specific embodiments of control sequencing templates andcontrol microparticles, it should be understood clearly that thesedescriptions are made only by way of example and are not intended tolimit the scope of the present teachings or claims. What has beendisclosed herein has been provided for the purposes of illustration anddescription. It is not intended to be exhaustive or to limit what isdisclosed to the precise forms described. Many modifications andvariations will be apparent to the practitioner skilled in the art. Whatis disclosed was chosen and described in order to best explain theprinciples and practical application of the disclosed embodiments of theart described, thereby enabling others skilled in the art to understandthe various embodiments and various modifications that are suited to theparticular use contemplated. It is intended that the scope of what isdisclosed be defined by the following claims and their equivalents.

What is claimed:
 1. A method for generating a circular double-strandedpolynucleotide, comprising: a) hybridizing a first double-strandednucleic acid adaptor having a first target-joining end and a firstoverhang end to a first single-stranded blocking oligonucleotide over atleast a portion of the length of the first overhang end, wherein thefirst overhang end contains a first overhang nucleic acid sequence, andwherein the sequence of the first single-stranded blockingoligonucleotide is complementary to at least a portion of the firstoverhang nucleic acid sequence; b) hybridizing a second double-strandednucleic acid adaptor having a second target joining end and a secondoverhang end to a second single-stranded blocking oligonucleotide overat least a portion of the length of the second overhang end, wherein (i)the second overhang end contains a second overhang nucleic acidsequence, (ii) the sequence of the second single-stranded blockingoligonucleotide is complementary to at least a portion of the secondoverhang nucleic acid sequence and (iii) the first and second overhangnucleic acid sequences are at least partially complementary to eachother; c) joining a linear double-stranded target polynucleotide havinga first end and a second end to the first double-stranded nucleic acidadaptor and the second double-stranded nucleic acid adaptor, wherein thefirst end is joined to the first target joining end of the firstdouble-stranded nucleic acid adaptor, and the second end is joined tothe second target joining end of the second double-stranded nucleic acidadaptor; and d) subjecting the linear double-stranded targetpolynucleotide that is joined to the first and second double-strandednucleic acid adaptors to conditions that decrease the stability of thehybridization between the first overhang end and the firstsingle-stranded blocking oligonucleotide and between the second overhangend and the second single-stranded blocking oligonucleotide such thatthe first overhang end of the first double-stranded nucleic acid adaptorand the second overhang end of the second double-stranded nucleic acidadaptor are exposed and the first overhang end of the firstdouble-stranded nucleic acid adaptor is hybridized to the secondoverhang end of the second double-stranded nucleic acid adaptor to forma double-stranded adaptor joining region and resulting in a nick at theend of each overhang end, thereby forming a circular double-strandedpolynucleotide having two nicks in the adaptor joining region.
 2. Themethod of claim 1, wherein the first and second overhang ends are 5′overhang ends or wherein the first and second overhang ends are 3′overhang ends.
 3. The method of claim 1, further comprising subjectingthe circular double-stranded polynucleotide to nick translation.
 4. Themethod of claim 3, wherein the nick translation is conducted in areaction mixture comprising (i) a coupled 5′ to 3′ DNApolymerization/degradation reaction mixture, or (ii) a coupled 5′ to 3′DNA polymerization/strand displacement reaction mixture.
 5. The methodof claim 3, wherein the nick translation is conducted in a reactionmixture comprising a DNA polymerase enzyme.
 6. The method of claim 3,wherein the nick translation is conducted in a reaction mixturecomprising an enzyme selected from the group consisting of E. coli DNApolymerase I, Taq DNA polymerase, Vent DNA polymerase, Klenow DNApolymerase I, Tfi DNA polymerase, Bst DNA polymerase, and phi29 DNApolymerase.
 7. The method of claim 3, wherein the nick translation isconducted in a reaction mixture comprising deoxyribonucleosidetriphosphates.
 8. The method of claim 1, wherein the firstdouble-stranded nucleic acid adaptor comprises biotin, or the seconddouble-stranded nucleic acid adaptor comprises biotin, or both the firstand second double-stranded nucleic acid adaptors comprise biotin.
 9. Themethod of claim 1, wherein the first single-stranded blockingoligonucleotide and/or the second single-stranded blockingoligonucleotide is shorter than the overhang end to which it hybridizes.10. The method of claim 3, further comprising cleaving the circulardouble-stranded polynucleotide with a single-strand specificendonuclease enzyme at the position of the nicks to release a linearpolynucleotide which includes a mate pair construct.
 11. The method ofclaim 1, wherein a nucleotide at the position of at least one of thenicks lacks a 5′ phosphate group.
 12. The method of claim 1, wherein theconditions that decrease the stability of the hybridization between thefirst overhang end and the first single-stranded blockingoligonucleotide and between the second overhang end and the secondsingle-stranded blocking oligonucleotide include altering the ionicstrength, pH and/or temperature of a medium in which the lineardouble-stranded target polynucleotide that is joined to the first andsecond double-stranded nucleic acid adaptors is contained.